Improved peptide pharmaceuticals for insulin resistance

ABSTRACT

Described herein are methods of syntheses and therapeutic uses of covalently modified peptides and/or proteins. The covalently modified peptides and/or proteins allow for improved pharmaceutical properties of peptide and protein-based therapeutics.

CROSS REFERENCE

This application is filed pursuant to 35 U.S.C. § 371 as a United StatesNational Phase Application of International Application Ser. No.PCT/USUS2012/038434, filed May 17, 2012, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 61/487,640, filed May 18,2011, and U.S. Provisional Patent Application Ser. No. 61/543,716, filedOct. 5, 2011, which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The increasing prevalence of diabetes mellitus is a world health crisisof epidemic proportions that is a major contributor to patient morbidityand mortality and a major economic burden. Obesity is an important riskfactor for type 2 diabetes, and roughly 90% of patients with type 2diabetes are overweight or obese. Obesity is a rapidly increasingproblem worldwide and currently more than 65% of adults in the U.S. areoverweight (Hedley, A. A., et al. (2004) JAMA 291: 2847-2850). There isa need for development of safe and efficacious pharmaceutical treatmentsfor obesity and diabetes mellitus.

SUMMARY OF THE INVENTION

Described herein are compositions and methods for treatment orprevention of disorders associated with the insulin resistance includingand not limited to obesity, the metabolic syndrome, type 2 diabetes,hypertension, atherosclerosis or the like. In some embodiments, themethods include prophylactic and/or therapeutic treatment with peptidesand/or proteins. Peptide and/or protein pharmaceuticals often sufferfrom several limitations in their use in medicine (Nestor, J. J., Jr.(2007) Comprehensive Medicinal Chemistry II 2: 573-601) short durationof action, poor bioavailability, and lack of receptor subtypeselectivity. In addition, peptides and/or proteins are unstable informulations, often being subject to aggregation.

Described herein are certain covalently modified peptides and/orproteins (for example, GLP-1, glucagon, related analogs or the like)that allow for longer duration of action and/or improved bioavailabilityupon administration of the modified peptides and/or proteins. Suchcovalently modified peptides and/or proteins are suitable for preventionand/or treatment of conditions associated with obesity, the metabolicsyndrome, insulin resistance, type 2 diabetes, hypertension,atherosclerosis, or the like.

In some embodiments, the covalently modified peptides and/or proteinsdescribed herein are attached to glycoside surfactants. In one aspect,the covalently modified peptides and/or proteins are attached to aglycoside surfactant wherein the peptide and/or protein is attached tothe glycoside in the surfactant and the glycoside is then attached to ahydrophobic group. Also provided herein, in some embodiments, arereagents and intermediates for synthesis of modified peptides and/orproteins (e.g., modified GLP-1, glucagon, analogs of glucagon or GLP-1,or the like) through the incorporation of surfactants.

Provided herein, in some embodiments, are peptide products comprising asurfactant X, covalently attached to a peptide, the peptide comprising alinker amino acid U and at least one other amino acid:

-   -   wherein the surfactant X is a group of Formula I:

-   -   -   wherein:            -   R^(1a) is independently, at each occurrence, a bond, H,                a substituted or unsubstituted C₁-C₃₀ alkyl group, a                substituted or unsubstituted alkoxyaryl group, or a                substituted or unsubstituted aralkyl group;            -   R^(1b), R^(1c), and R^(1d) are each, independently at                each occurrence, a bond, H, a substituted or                unsubstituted C₁-C₃₀ alkyl group, a substituted or                unsubstituted alkoxyaryl group, or a substituted or                unsubstituted aralkyl group;            -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,                —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or                —CH₂—S—;            -   W² is —O—, —CH₂— or —S—;            -   R² is independently, at each occurrence, a bond, H, a                substituted or unsubstituted C₁-C₃₀ alkyl group, a                substituted or unsubstituted alkoxyaryl group, or a                substituted or unsubstituted aralkyl group, —NH₂, —SH,                C₂-C₄-alkene, C₂-C₄-alkync, —NH(C═O)—CH₂—Br,                —(CH₂)_(m)-maleimide, or —N₃;            -   n is 1, 2 or 3; and            -   m is 1-10;

    -   the peptide is selected from Formula II:

Formula II (SEQ. ID. NO. 1)aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-aa₃₀-aa₃₁-aa₃₂-aa₃₃-aa₃₄- aa₃₅-aa₃₆-aa₃₇-Z

-   -   -   wherein:            -   Z is OH, or —NH—R³, wherein R³ is H or C₁-C₁₂                substituted or unsubstituted alkyl,            -   or a PEG chain of less than 10 Da;            -   aa₁ is His, N-Ac-His, pGlu-His, or N—R³-His;            -   aa₂ is Ser, Ala, Gly, Aib, Ac4c or Ac5c;            -   aa₃ is Gln, or Cit;            -   aa₄ is Gly, or D-Ala;            -   aa₅ is Thr, or Ser;            -   aa₆ is Phe, Trp, F2Phe, Me2Phe, or Nal2;            -   aa₇ is Thr, or Ser;            -   aa₈ is Ser, or Asp;            -   aa₉ is Asp, or Glu;            -   aa₁₀ is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO;            -   aa₁₁ is Ser, Asn, or U;            -   aa₁₂ is Lys, Glu, Ser, Arg, or U;            -   a₁₃ is absent or Tyr, Gln, Cit, or U;            -   aa₁₄ is absent or Leu, Met, Nle, or U;            -   aa₁₅ is absent or Asp, Glu, or U;            -   aa₁₆ is absent or Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or                U;            -   aa₁₇ is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c,                Ac5c, or U;            -   aa₁₈ is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U;            -   aa₁₉ is absent or Ala, Val, Aib, Ac4c, Ac5c, or U;            -   aa₂₀ is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c,                Ac5c, or U;            -   aa₂₁ is absent or Asp, Glu, Leu, Aib, Ac4c Ac5c, or U;            -   aa₂₂ is absent or Phe, Trp, Nal2, Aib, Ac4c, Ac5c, or U            -   aa₂₃ is absent or Val, Ile, Aib, Ac4c, Ac5c, or U;            -   aa₂₄ is absent or Gln, Ala, Glu, Cit, or U;            -   aa₂₅ is absent or Trp, Nal2, or U;            -   aa₂₆ is absent or Leu, or U;            -   aa₂₇ is absent or Met, Val, Nle, Lys, or U;            -   aa₂₈ is absent or Asn, Lys, or U;            -   aa₂₉ is absent or Thr, Gly, Aib, Ac4c, Ac5c, or U;            -   aa₃₀ is absent or Lys, Aib, Ac4c, Ac5c, or U;            -   aa₃₁ is absent or Arg, Aib, Ac4c, Ac5c, or U;            -   aa₃₂ is absent or Asn, Aib, Ac4c, Ac5c, or U;            -   aa₃₃ is absent or Arg, Aib, Ac4c, Ac5c, or U;            -   aa₃₄ is absent or Asn, Aib, Ac4c, Ac5c, or U;            -   aa₃₅ is absent or Asn, Aib, Ac4c, Ac5c, or U;            -   aa₃₆ is absent or lie, Aib, Ac4c, Ac5C, or U;            -   aa₃₆ is absent or Ala, Aib, Ac4c, Ac5C, or U;            -   a₃₇ absent or U;            -   U is a natural or unnatural amino acid comprising a                functional group used for covalent attachment to the                surfactant X;

    -   wherein any two of aa₁-aa₃₇ are optionally cyclized through        their side chains to form a lactam linkage; and

    -   provided that one, or at least one of aa₁₁-aa₃₇ is the linker        amino acid U covalently attached to X.

In some embodiments, n is 1. In some embodiments, n is 2, and a firstglycoside is attached to a second glycoside via bond between W² of thefirst glycoside and any one of OR^(1b), OR^(1c) or OR^(1d) of the secondglycoside. In some embodiments, n is 3, and a first glycoside isattached to a second glycoside via bond between W² of the firstglycoside and any one of OR^(1b), OR^(1c) or OR^(1d) of the secondglycoside, and the second glycoside is attached to a third glycoside viabond between W² of the second glycoside and any one of OR^(1b), OR^(1c)or OR^(1d) of the third glycoside.

In one embodiment, compounds of Formula I-A are compounds wherein X hasthe structure:

-   -   wherein:        -   R^(1a) is H, a protecting group, a substituted or            unsubstituted C₁-C₁₀ alkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, H, a protecting group, or a substituted or            unsubstituted C₁-C₃₀ alkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C—O), —(C—O)—O—, —(C—O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W is —O—, —S—;        -   R² is a bond, C₂-C₄-alkene, C₂-C₄-alkyne, or            —(CH₂)_(m)-maleimide; and        -   m is 1-10.

In another embodiment, compounds of Formula I-A are compounds wherein Xhas the structure:

Accordingly, in the embodiment described above, R² is a bond.

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —C(═O)NH—, R² is a bond between W¹ and an amino acidresidue U within the peptide (e.g., an amino group in the sidechain of alysine residue present in the peptide).

In a further embodiment, compounds of Formula I-A are compounds whereinX has the structure:

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —CH₂— and R² is an alkyl-linked maleimide functional groupon X and R² is attached to a suitable moiety of an amino acid residue Uwithin the peptide (e.g., a thiol group in a cysteine residue of thepeptide forms a thioether with the maleimide on X).

In yet another embodiment, compounds of Formula I-A are compoundswherein X has the structure:

-   -   wherein:        -   R^(1a) is H, a protecting group, a substituted or            unsubstituted C₁-C₃₀ alkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, H, a protecting group, or a substituted or            unsubstituted C₁-C₃₀ alkyl group;        -   W¹ is —(C═O)—NH—;        -   W² is —O—;        -   R² is a bond.

In an additional embodiment, compounds of Formula I-A are compoundswherein X has the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In some embodiments described above and herein, R^(1a) is a substitutedor unsubstituted C₁-C₃₀ alkyl group.

In some embodiments described above and herein, R^(1a) is a substitutedor unsubstituted C₆-C₂₀ alkyl group.

Also contemplated herein are alternate embodiments wherein X in FormulaI-A has the structure:

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —S—, R² is a C₁-C₃₀ alkyl group, W² is S, R^(1a) is a bondbetween W² and a suitable moiety of an amino acid residue U within thepeptide (e.g., a thiol group in a cysteine residue of the peptide formsa thioether with X).

In another exemplary embodiment of the structure of X described above,W¹ is —O—, R² is a C₁₋₃₀ alkyl group, W² is O, R^(1a) is a bond betweenW² and a suitable moiety of an amino acid residue U within the peptide(e.g., a hydroxyl group in a serine or threonine residue of the peptideforms an ether with X).

In some embodiments, U is used for covalent attachment to X and is adibasic natural or unnatural amino acid, a natural or unnatural aminoacid comprising a thiol, an unnatural amino acid comprising a N₃ group,an unnatural amino acid comprising an acetylenic group, or an unnaturalamino acid comprising a —NH—C(—O)—CH₂—Br or a —(CH₂)m-maleimide, whereinm is 1-10.

In some embodiments of the peptide product, the surfactant is a 1-alkylglycoside class surfactant. In some embodiments of the peptide product,the surfactant is attached to the peptide via an amide bond.

In some embodiments of the peptide product, the surfactant X iscomprised of 1-eicosyl beta-D-glucuronic acid, 1-octadecylbeta-D-glucuronic acid, 1-hexadecyl beta-D-glucuronic acid,1-tetradecylbeta D-glucuronic acid, 1-dodecyl beta D-glucuronic acid,1-decyl beta-D-glucuronic acid, 1-octyl bcta-D-glucuronic acid,1-cicosyl beta-D-diglucuronic acid, 1-octadecyl beta-D-diglucuronicacid, 1-hexadecyl beta-D-diglucuronic acid, 1-tetradecylbeta-D-diglucuronic acid, 1-dodecyl beta-D-diglucuronic acid, 1-decylbeta-D-diglucuronic acid, 1-octyl beta-D-diglucuronic acid, orfunctionalized 1-ecosyl beta-D-glucose, 1-octadecyl beta-D-glucose,1-hexadecyl beta-D-glucose, 1-tetradecyl beta-D-glucose, 1-dodecylbeta-D-glucose, 1-decyl beta-D-glucose, 1-octyl beta-D-glucose,1-eicosyl beta-D-maltoside, 1-octadecyl beta-D-maltoside, 1-hexadecylbcta-D-maltoside, 1-dodecyl bcta-D-maltoside, 1-decyl beta-D-maltoside,1-octyl beta-D-maltoside, and the like, and the peptide product isprepared by formation of a linkage between the aforementioned groups anda group on the peptide (e.g., a —COOH group in the aforementioned groupsand an amino group of the peptide).

In some embodiments of the peptide product, U is a terminal amino acidof the peptide. In some embodiments of the peptide product, U is anon-terminal amino acid of the peptide. In some embodiments of thepeptide product, U is a natural D- or L-amino acid. In some embodimentsof the peptide product, U is an unnatural amino acid. In someembodiments of the peptide product, U is selected from Lys, Cys, Orn, oran unnatural amino acid comprising a functional group used for covalentattachment to the surfactant X.

In some embodiments of the peptide product, the functional group usedfor covalent attachment of the peptide to the surfactant X is —NH₂, —SH,—OH, —N₃, haloacetyl, a —(CH₂)_(m)-maleimide (wherein m is 1-10), or anacetylenic group.

In some embodiments side chain functional groups of two different aminoacid residues are linked to form a cyclic lactam. For example, in someembodiments, a Lys side chain forms a cyclic lactam with the side chainof Glu. In some embodiments such lactam structures are reversed and areformed from a Glu and a Lys. Such lactam linkages in some instances areknown to stabilize alpha helical structures in peptides (Condon, S. M.,et al. (2002) Bioorg Med Chem 10: 731-736; Murage, E. N., et al (2008)Bioorg Med Chem 16: 10106-12); Murage, E. N., et al. (2010) J Med Chem53: 6412-20). In some embodiments cysteine residues may be linkedthrough disulfide formation in order to accomplish a similar form ofconformational restriction and assist in the formation of helicalstructures (Li, Y., et al. (2011) Peptides 32: 1400-1407. In someembodiments side chain functional groups of two different amino acidresidues are linked to form a heterocycle generated through a “clickreaction” between side chain azide and alkyne functional groups in orderto achieve a similar form of conformational restriction and stabilizedhelical conformations (Le Chevalier Isaad A., et al. (2009) J PeptideSci 15: 451-4).

In some embodiments, the peptide product comprising a covalently linkedalkyl glycoside is a covalently modified glucagon or analog thereof. Insome of such embodiments, the peptide product contains a covalentlylinked 1-O-alkyl β-D-glucuronic acid and the peptide is an analog ofglucagon.

In some embodiments, a peptide product comprising a covalently linkedalkyl glycoside is a covalently modified GLP-1, or analog thereof. Insome of such embodiments, the peptide product comprises a covalentlylinked 1-O-alkyl β-D-glucuronic acid and the peptide is an analog ofGLP-1.

In some embodiments, the peptide product of Formula I-A has thestructure of Formula III-A

Formula III-A (SEQ. ID. NO. 2)aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-Z

-   -   wherein:    -   Z is OH, or —NH—R³, wherein R³ is H, or C₁-C₁₂ substituted or        unsubstituted alkyl, or a PEG chain of less than 10 Da;    -   aa₁ is His, N—Ac-His, pGlu-His, or N—R³-His;    -   aa₂ is Ser, Ala, Gly, Aib, Ac4c, or Ac5c;    -   aa₃ is Gln, or Cit;    -   aa₄ is Gly, or D-Ala;    -   aa₅ is Thr, or Ser,    -   aa₆ is Phe, Trp, F2Phe, Me2Phe, or Nal2;    -   aa₇ is Thr, or Ser;    -   aa₈ is Ser, or Asp;    -   aa₉ is Asp, or Glu;    -   aa₁₀ is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO;    -   aa₁₁ is Ser, Asn, or U;    -   aa₁₂ is Lys, Glu, Ser, Arg, or U(X);    -   aa₁₃ is absent or Tyr, Gln, Cit, or U(X);    -   aa₁₄ is absent or Leu, Met, Nle, or U(X);    -   aa₁₅ is absent or Asp, Glu, or U(X);    -   aa₁₆ is absent or Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or U(X);    -   aa₁₇ is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c, or        U(X);    -   aa₁₈ is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X);    -   aa₁₉ is absent or Ala, Val, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₀ is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c, or        U(X);    -   aa₂₁ is absent or Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₂ is absent or Phe, Trp, Nal2, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₃ is absent or Val, Ile, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₄ is absent or Gln, Ala, Glu, Cit, or U(X);    -   aa₂₅ is absent or Trp, Nal2, or U(X);    -   aa₂₆ is absent or Leu, or U(X);    -   aa₂₇ is absent or Met, Val, Nle, Lys, or U(X);    -   aa₂₈ is absent or Asn, Lys, or U(X);    -   aa₂₉ is absent or Thr, Gly, Aib. Ac4c, Ac5c, or U(X);    -   wherein any two of aa₁-aa₂₉ are optionally cyclized through        their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₆, aa₁₇, a₁₈, aa₁₉,        aa₂₀, aa₂₁, aa₂₂, aa₂₃, aa₂₄, aa₂₅, aa₂₆, aa₂₇, aa₂₈ or a₂₉ is        the natural or unnatural amino acid U covalently attached to X.

In some embodiments, the peptide product of Formula I-A has thestructure of Formula III-B:

Formula III-B (SEQ. ID. NO. 3)His₁-aa₂-aa₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂- aa₂₃-Z

-   -   wherein:        -   Z is OH, or —NH—R³, wherein R³ is H or substituted or            unsubstituted C₁-C₁₂ alkyl; or a PEG chain of less than 10            Da;            -   aa₂ is Ser, Ala, Gly, Aib, Ac4c, or Ac5c;            -   aa₃ is Gln, or Cit;            -   aa₆ is Phe, Trp, F2Phe, Me2Phe, MePhe, or Nal2;            -   aa₁₀ is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO;            -   aa₁₁ is Ser, Asn, or U(X);            -   aa₁₂ is Lys, Glu, Ser or U(X);            -   aa₁₃ is absent or Tyr, Gln, Cit, or U(X);            -   aa₁₄ is absent or Leu, Met, Nle, or U(X);            -   aa₁₅ is absent or Asp, Glu, or U(X);            -   aa₁₆ is absent or Ser, Gly, Glu, Aib, Ac4c, Ac5c, Lys,                R, or U(X);            -   aa₁₇ is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c,                Ac5c, or U(X);            -   aa₁₈ is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or                U(X);            -   aa₁₉ is absent or Ala, Val, Aib, Ac4c, Ac5c, or U(X);            -   aa₂₀ is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c,                Ac5c, or U(X);            -   aa₂₁ is absent or Asp, Glu, Leu, Aib, Ac4c, Ac5c, or                U(X);            -   aa₂₂ is absent or Phe, Aib, Ac4c, Ac5c, or U(X)            -   aa₂₃ is absent or Val, Ile, Aib, Ac4c, Ac5c, or U(X);        -   wherein any two of aa₁-aa₂₃ are optionally cyclized through            their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₆, aa₁₇, a₁₈, aa₁₉,        aa₂₀, aa₂₁, aa₂₂, aa₂₃ or aa₂₄ is the natural or unnatural amino        acid U covalently attached to X.

In some embodiments of Formula I-A, III-A, III-B or Formula V, U is anylinker amino acid described herein.

In some embodiments of Formula I-A, III-A, II-B or Formula V, aa₁₂ islysine. In some embodiments of Formula I-A. III-A, III-B or Formula V,aa₁₄ is leucine.

In some embodiments of Formula I-A, III-A, III-B or Formula V, aa₁₈ is alysine residue attached to X.

In some embodiments of Formula I-A, III-A, III-B or Formula V, aa₁₇ is ahomo Arginine (hArg) residue.

In some embodiments of Formula I-A, III-A, III-B or Formula V, aa₁₇ is aglycine residue.

In some embodiments of Formula I-A, III-A, III-B or Formula V, aa₂ is anAib or Ac4c residue.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide comprises one or more Aib residues.

In some embodiments of Formula I-A, III-A, III-B or Formula V, peptidecomprises one or more Aib residues at the C-terminus.

In some embodiments of Formula I-A, III-A, II-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 318) His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-aa₁₇-Lys(N-omega- 1′-alkyl beta-D-glucuronyl)₁₈-aa₁₉-NH₂;

-   -   wherein    -   aa₂ is Aib or Ac4c;    -   aa₁₇ is Arg, hArg or Gln;    -   aa₁₉ is Aib, Ac4c or Ac5c; and    -   alkyl is a C₈ to C₂₀ linear alkyl chain.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product ha the structure:

(SEQ. ID. NO. 319) His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-aa₁₇-Lys(N-omega- 1′-alkyl beta-D-glucuronyl)₁₈-aa₁₉-aa₂₀-NH₂;wherein

-   -   aa₂ is Aib or Ac4c,    -   aa₁₇ is Arg, hArg or Gln,    -   aa₁₉ and aa20 are individually Aib, Ac4c or Ac5c; and    -   alkyl is a C₈ to C₂₀ linear alkyl chain.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product ha the structure:

(SEQ. ID. NO. 320) His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-Lys(N-omega-1′-alkyl beta-D-glucuronyl)₁₈-aa₁₉-NH₂;

-   -   wherein    -   aa₂ is Aib or Ac4c;    -   aa₁₆ is Aib or Ac4c;    -   aa₁₇ is Arg, hArg or Gln;    -   aa₁₉ is Aib, Ac4c or Ac5c; and    -   alkyl is a C₈ to C₂₀ linear alkyl chain.

In some embodiments of Formula I-A, III-A, III-B or Formula V, aa₁₆ andaa₂₀ are cyclized to form a lactam linkage.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 321) His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇ Ala₁₈-Ala₁₉-aa₂₀-Glu₂₁-Phe₂₂-Ile₂₃-Lys(N-omega-1′-alkyl beta-D-glucuronyl)₂₄-Trp₂₅-Leu₂₆-aa₂₇-Asn₂₈-Thr₂₉-NH₂;wherein

-   -   aa₂ is Aib or Ac4c;    -   aa₁₆ and aa₂₀ are each individually either Lys or Glu and are        cyclized through their side chains to form a lactam linkage;    -   aa₁₇ is Arg, hArg or Gln;    -   aa₂₇ is Met or Nle; and    -   alkyl is a C₈-C₂₀ linear alkyl chain.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 322)His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈₋Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-cyclic(Glu₁₆-Gln₁₇-Ala₁₈-Ala₁₉-Lys₂₀)-Glu₂₁-Phe₂₂-Ile₂₃-Lys(N-omega-1′-alkyl beta-D-glucuronyl)₂₄-Trp₂₅-Leu₂₆-Met₂₇-Asn₂₈-aa₂₉-NH₂;wherein aa₂ is Aib or Ac4c, aa29 is Thr, Aib, Ac4c, or Ac5c, and the1′-alkyl group is selected from dodecyl, tetradecyl, hexadecyl, oroctadecyl; and the side chains on the amino acids in position 16 and 20are cyclized to form a side chain lactam.

In some embodiments of Formula I-A, III-A, III-B or Formula V, aa₁₂ andaa₁₆ are cyclized to form a lactam linkage.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 323) His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-aa₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-Lys(N-omega-1′-alkyl beta-D-glucuronyl)₁₈-aa₁₉-aa₂₀-NH₂;

-   -   wherein    -   aa2 is Aib or Ac4c;    -   aa₁₂ and aa₁₆ are each individually either Lys or Glu and are        cyclized through their side chains to form a lactam linkage;    -   aa₁₇ is Arg, hArg;    -   aa₁₉ and aa₂₀ are individually either Aib, Ac4c or Ac5c; and    -   alkyl is a C₈-C₂₀ linear alkyl chain.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 324) His₁-Ac4c₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-cyclo(Glu₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Lys₁₆)-aa₁₇-Lys(N-omega-1′-alkyl beta-D-glucuronyl)₁₈-Aib₁₉-Aib₂₀-NH₂;wherein

-   -   aa₁₂ and aa₁₆ are cyclized through their side chains to form a        lactam linkage;    -   aa₁₇ is Arg or hArg; and    -   alkyl is a C₁₂, C₁₄, C₁₆, or C₁₈ linear alkyl chain.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 325) His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-aa₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-Lys(N-omega-1′-alkyl beta-D-glucuronyl)₁₈-aa₁₉-aa₂₀-NH₂;

-   -   wherein        -   aa₁₂ and aa₁₆ are each individually either Lys or Glu        -   and aa₁₂ and aa₁₆ are cyclized through their side chains to            form a lactam linkage;        -   aa₁₇ is Arg or hArg; aa₁₉ and aa₂₀ are individually either            Aib, Ac4c or Ac5c; and the        -   1′-alkyl group is selected from dodecyl, tetradecyl,            hexadecyl, or octadecyl.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 326)His₁-aa₂-Gln₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Aib₁₇-Lys(N-omega-1′-dodecyl beta-D-glucuronyl)₁₈-aa₁₉-NH₂;

-   -   wherein aa₂ is Aib or Ac4c, aa₆ is Me2Phe, MePhe, or Phe; and        aa₁₉ is Aib, Ac4c, or Ac5c.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 327)His₁-aa₂-Gln₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-aa₁₇-Lys(N-omega-1′-dodecyl beta-D-glucuronyl)₁₈-aa₁₉₋aa₂₀-NH₂;wherein aa₂ is Aib or Ac4c, aa₆ is Me2Phe, MePhe, or Phe; aa₁₇ is Arg orhArg, and aa₁₉ or aa₂₀₀ is Aib, Ac4c, or Ac5c.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 328) His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-cyclo(Glu₁₆-Arg₁₇-Ala₁₈-Ala₁₉-Lys₂₀)-(N-omega-1′-alkyl beta-D-glucuronyl)₂₁-Phe₂₂-aa₂₃-NH₂;wherein aa₂₃ is Aib, Ac4c, or Ac5c and the 1′-alkyl group is selectedfrom dodecyl, tetradecyl, hexadecyl, or octadecyl.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 329)His₁-aa₂-Gln₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-aa₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-aa₁₈-Ala₁₉-aa₂₀-Lys(N-omega-1′-alkyl beta-D-glucuronyl)₂₁-Phe₂₂-aa₂₃-NH₂;

-   -   wherein        -   aa₂ is Aib or Ac4c;        -   aa₆ is Me2Phe. MePhe, or Phe;        -   aa₁₂ and aa₁₆ are each individually either Lys or Glu;        -   and aa₁₆ and aa₂₀ are cyclized through their side chains to            form a lactam linkage;        -   aa₁₇ is Arg, hArg or Gln;        -   aa₁₈ is Aib or Ala;        -   aa₂₃ is Aib, Ac4c, or Ac5c and the 1′-alkyl group is            selected from dodecyl, tetradecyl, hexadecyl, or octadecyl.

In some embodiments of Formula I-A, III-A, III-B or Formula V, thepeptide product has the structure:

(SEQ. ID. NO. 330)His₁-aa₂-Gln₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-aa₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-Lys(N-omega-1′-alkylbeta-D-glucuronyl)₁₈-aa₁₉-aa₂₀-NH₂;

-   -   wherein        -   aa₂ is Aib or Ac4c;        -   aa₆ is Phe;        -   aa₁₂ and aa₁₆ are each individually either Lys or Glu; and            aa₁₇ and aa₁, are cyclized through their side chains to form            a lactam linkage;        -   aa₁₇ is Arg or hArg;        -   aa₁₉ is Aib, Ac4c, or Ac5c;        -   aa₂₀ is Aib, Ac4c, or Ac5c and the and the 1′-alkyl group is            selected from dodecyl, tetradecyl, hexadecyl, or octadecyl.

In some embodiments, for any compound of Formula I-A, Formula III-A,Formula III-B or Formula V, X is comprised of a dodecyl alkyl chain.

In some embodiments, the peptide product is a biologically activepeptide product that binds to the GLP1R and/or to the GLCR.

In a specific embodiment, the peptide products of Formula I-A, III-A,III-B or Formula V, described above and herein have the followingstructure:

wherein R^(1a) is a C₁-C₂₀ alkyl chain as described in Table 1 of FIG.1, R′ is a peptide as described in Table 1 of FIG. 1 and Table 2 of FIG.2, W² of Formula I-A is —O—, and W¹ of Formula I-A is —(C═O)NH— and ispart of an amide linkage to the peptide R′. In some of such embodiments,R^(1a) is a C₆-C₂₀ alkyl chain. In some of such embodiments, R^(1a) is aC₈-C₂₀ alkyl chain. In some of such embodiments, R^(1a) is a C₁₂-C₂₀alkyl chain. In some of such embodiments, R^(1a) is a C₁₂-C₁₆ alkylchain.

In embodiments described above, an amino moiety of an amino acid and/ora peptide R′ (e.g., an amino group of an amino acid residue such as aLysine, or a lysine residue within the peptide R′) is used to form acovalent linkage with a compound of structure:

wherein R^(1a) is a C₁-C₂₀ alkyl chain as described above and in Table 1of FIG. 1 and Table 2 of FIG. 2.

In such cases, the amino acid residue having an amino moiety (e.g., aLysine within the peptide R′) which is used to form a covalent linkageto the compound A described above, is a linker amino acid U which isattached to a surfactant X having the structure of Formula A.Accordingly, as one example, Lys(C12) of Table 1 of FIG. 1 or Table 2 ofFIG. 2 has the following structure:

Also contemplated within the scope of the embodiments presented hereinare peptide products of Formula I-A derived from maltouronic acid-basedsurfactants through binding at either or both carboxylic acid functions.Thus, as one example, peptides in Table 1 of FIG. 1 or Table 2 of FIG. 2comprise a lysine linker amino acid bonded to a maltouronic acid basedsurfactant X and having a structure:

It will be understood that in one embodiment, compounds of Formula I-Aare prepared by attaching a lysine to a group X, followed by attachmentof additional amino acid residues and/or peptides are attached to thelysine-X compound to obtain compounds of Formula I-A. It will beunderstood that other natural or non-natural amino acids describedherein are also suitable for attachment to the surfactant X and aresuitable for attaching additional amino acid/peptides to obtaincompounds of Formula I-A. It will be understood that in anotherembodiment, compounds of Formula I-A are prepared by attaching a fulllength or partial length peptide to a group X, followed by optionalattachment of additional amino acid residues and/or peptides areattached to obtain compounds of Formula I-A.

In a specific embodiment, provided herein is a compound selected fromcompounds of Table 1 of FIG. 1 or Table 2 of FIG. 2.

Also provided herein are pharmaceutical compositions comprising atherapeutically effective amount of a peptide product described above,or acceptable salt thereof, and at least one pharmaceutically acceptablecarrier or excipient.

In some embodiments of the pharmaceutical compositions, the carrier isan aqueous-based carrier. In some embodiments of the pharmaceuticalcompositions, the carrier is a nonaqueous-based carrier. In someembodiments of the pharmaceutical compositions, the nonaqueous-basedcarrier is a hydrofluoroalkane-like solvent that may comprise sub-micronanhydrous α-lactose or other excipients.

Contemplated within the scope of embodiments presented herein is thereaction of an amino acid and/or a peptide comprising a linker aminoacid U bearing a nucleophile, and a group X comprising a bearing aleaving group or a functional group that can be activated to contain aleaving group, for example a carboxylic acid, or any other reactinggroup, thereby allowing for covalent linkage of the amino acid and/orpeptide to a surfactant X via the linker amino acid U to provide apeptide product of Formula I-A.

Also contemplated within the scope of embodiments presented herein isthe reaction of an amino acid and/or a peptide comprising a linker aminoacid U bearing a bearing a leaving group or a functional group that canbe activated to contain a leaving group, for example a carboxylic acid,or any other reacting group, and a group X comprising a nucleophilicgroup, thereby allowing for covalent linkage of the amino acid and/orpeptide to a surfactant X via the linker amino acid U to provide apeptide product of Formula I-A.

It will be understood that, in one embodiment, Compounds of Formula I-Aare prepared by reaction of a linker amino acid U with X, followed byaddition of further residues to U to obtain the peptide product ofFormula I-A. It will be understood that in an alternative embodiment,Compounds of Formula I-A are prepared by reaction of a suitable peptidecomprising a linker amino acid U with X, followed by optional additionof further residues to U, to obtain the peptide product of Formula I-A.

Further provided herein are methods for synthesizing peptide productsdescribed above, comprising sequential steps of

-   -   (a) Coupling a peptide with an intermediate, i.e., a compound of        Formula IV:

-   -   wherein:    -   R^(1a) is independently, at each occurrence, a bond, H, a        leaving group, a protecting group, a natural or unnatural amino        acid, a substituted or unsubstituted C₁-C₃₀ alkyl group, a        substituted or unsubstituted alkoxyaryl group, or a substituted        or unsubstituted aralkyl group;    -   R^(1b), R^(1c), and R^(1d) are each independently, at each        occurrence, a bond, H, a leaving group, a protecting group, a        reversibly protected natural or unnatural amino acid, a        substituted or unsubstituted C₁-C₃₀ alkyl group, a substituted        or unsubstituted alkoxyaryl group, or a substituted or        unsubstituted aralkyl group;    -   W¹ is —CH₂—, —CH₂—O—, —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—,        —(C═S)—NH—, or —CH₂—S—;    -   W² is —O—, —CH₂— or —S—;    -   R² is independently, at each occurrence, a bond, H, a leaving        group, a protecting group, a reversibly protected natural or        unnatural amino acid, a substituted or unsubstituted C₁-C₃₀        alkyl group, a substituted or unsubstituted alkoxyaryl group, or        a substituted or unsubstituted aralkyl group, —NH₂, —SH,        C₂-C₄-alkene, C₂-C₄-alkyne, —NH(C═O)—CH₂—Br,        —(CH₂)_(m)-maleimide, or —N₃;    -   n is 1, 2 or 3;    -   m is 1-10;    -   and    -   (b) optionally deprotecting the coupled peptide of step (a).

In some embodiments of the methods, each natural or unnatural amino acidis independently, at each occurrence, a reversibly protected linkeramino acid. In some embodiments of the methods, each natural orunnatural amino acid is independently, at each occurrence, a reversiblyprotected or free lysine.

In some embodiments of the methods, the peptide is a peptide of FormulaII as described above.

In some embodiments of the methods,

-   -   n is 1;    -   W¹ is —(C═O)—;    -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group, a        substituted or unsubstituted 1-alkoxyaryl group, or a        substituted or unsubstituted 1-aralkyl group,    -   R² is a reversibly-protected lysine of D- or L-configuration.

In some embodiments of the methods,

-   -   n is 1;    -   W¹ is —(C═O)—;    -   R^(1a) is a substituted or unsubstituted C₈-C₃₀ alkyl group, a        substituted or unsubstituted 1-alkoxyaryl group, or a        substituted or unsubstituted 1-aralkyl group,    -   R² is a reversibly protected lysine of D- or L-configuration.

In some embodiments of the methods, R^(1a) is an octyl, decyl, dodecyl,tetradecyl, or hexadecyl group.

In some embodiments of the methods,

-   -   n is 1;    -   W¹ is —(C═O)—NH— or —(C═O)—O—;    -   R² is a substituted or unsubstituted C₁-C₃₀ alkyl hydrophobic        group, a substituted or unsubstituted 1-alkoxyaryl group, or a        substituted or unsubstituted 1-aralkyl group,    -   R^(1a) is a reversibly protected serine or threonine of D- or        L-configuration.

In some embodiments of the methods, R² is an octyl, decyl, dodecyl,tetradecyl or hexadecyl group.

In some embodiments of the methods,

-   -   n is 1;    -   m is 1-6;    -   W¹ is —CH₂—;    -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl        hydrophobic group, a substituted or unsubstituted 1-alkoxyaryl        group, or a substituted or unsubstituted 1-aralkyl group,    -   R² is —N₃, NH₂, —C₂-alkyne, —(CH₂)_(m)-maleimide,        NH—(C═O)—CH₂—Br, or NH—(C═O)—CH₂—;

In some embodiments of Formula IV,

-   -   n is 1;    -   W¹ is —(C═O)—O—;    -   R² is H,    -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl        hydrophobic group.

In some embodiments of the methods, W¹ is —(CH₂)O. In some embodimentsof the methods, n is 1. In some embodiments of the methods, n is 2, anda first glycoside is attached to a second glycoside via bond between W²of the first glycoside and any one of OR^(1b), OR^(1c) or OR^(1d) of thesecond glycoside.

In some embodiments of the methods, n is 3, and a first glycoside isattached to a second glycoside via bond between W² of the firstglycoside and any one of OR^(1b), OR^(1c) or OR^(1d) of the secondglycoside, and the second glycoside is attached to a third glycoside viabond between W² of the second glycoside and any one of OR^(1b), OR^(1c)or OR^(1d) of the third glycoside.

In some embodiments of the methods, the compound of Formula IV is areversibly protected N-ε-(1′-alkyl glucuronyl)-lysine of the D- orL-configuration, wherein R^(1a) is a substituted or unsubstituted C₁-C₂₀alkyl chain, a substituted or unsubstituted 1-alkoxyaryl group, or asubstituted or unsubstituted 1-aralkyl group.

In some embodiments of the methods, the compound of Formula TV is areversibly protected N-ε-(1′-dodecyl β-D-glucuronyl)-lysine of the D- orL-configuration.

In some embodiments of the methods, the deprotecting comprises the useof mild acid and or mild base treatments. In some embodiments of themethods, the deprotecting comprises the use of strong acids.

In some embodiments, the methods further comprise the steps ofchromatography, desalting of intermediates by reversed-phase,high-performance liquid chromatography or ion exchange chromatography ofintermediates.

A pharmaceutical composition comprising a therapeutically effectiveamount of a peptide product described above and herein, or acceptablesalt thereof, and at least one pharmaceutically acceptable carrier orexcipient.

Provided herein is a method for treating a condition associated withinsulin resistance comprising administration of any compound describedherein to an individual in need thereof.

Provided herein are methods for treating diabetes, diabetic retinopathy,diabetic neuropathy, diabetic nephropathy, wound healing, insulinresistance, hyperglycemia, hyperinsulinemia, metabolic syndrome,diabetic complications, elevated blood levels of free fatty acids orglycerol, hyperlipidemia, obesity, hypertriglyceridemia,atherosclerosis, acute cardiovascular syndrome, infarction, ischemicreperfusion or hypertension, comprising administering a therapeuticallyeffective amount of a peptide product described above and herein to anindividual in need thereof.

Provided herein are methods of reducing weight gain or inducing weightloss comprising administering to a subject in need thereof atherapeutically effective amount of a peptide product described aboveand herein to an individual in need thereof.

Provided herein are methods for treating mammalian conditionscharacterized by obesity-linked insulin resistance or the metabolicsyndrome comprising administering to a subject in need thereof a weightloss-inducing or insulin-sensitizing amount of a peptide productdescribed above and herein to an individual in need thereof.

In some embodiments, the condition to be treated is the metabolicsyndrome (Syndrome X). In some embodiments, the condition to be treatedis diabetes. In some embodiments, the condition to be treated ishyperlipidemia. In some embodiments, the condition to be treated ishypertension. In some embodiments, the condition to be treated isvascular disease including atherosclerosis, or the systemic inflammationcharacterized by elevated C reactive protein.

In some embodiments of the methods, the effective amount of the peptideproduct for administration is from about 0.1 μg/kg/day to about 100.0μg/kg/day, or from 0.01 μg/kg/day to about 1 mg/kg/day or from 0.1μg/kg/day to about 50 mg/kg/day. In some embodiments, the peptideproduct is administered parenterally. In some embodiments, the peptideproduct is administered subcutaneously. In some embodiments, the methodof administration of the peptide product is nasal insufflation.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular subject in need of treatment maybe varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andduration of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and the hostundergoing therapy.

Provided herein are methods of treating the metabolic syndrome, or itscomponent diseases, comprising administering to a subject in needthereof a therapeutically effective amount of a peptide productdescribed above. In some embodiments, the metabolic syndrome conditionhas progressed to diabetes.

Also provided herein is a covalently modified GLCR and/or GLP1R bindingpeptide or analog thereof, comprising a hydrophilic group as describedherein; and a hydrophobic group covalently attached to the hydrophilicgroup. In specific embodiments, the covalently modified peptide and/orprotein product comprises a hydrophilic group that is a saccharide and ahydrophobic group that is a C₁-C₂₀ alkyl chain or an aralkyl chain.

In one embodiment, provided is a method for chemically modifying amolecule by covalent linkage to a surfactant to increase or sustain thebiological action of the composition or molecule, for example, receptorbinding or enzymatic activity. In some embodiments, the molecule is apeptide. The method additionally can include further modificationcomprising covalent attachment of the molecule in the composition to apolymer such as polyethylene glycol.

In another embodiment, provided is a method of reducing or eliminatingimmunogenicity of a peptide and/or protein drug by covalently linkingthe peptide chain to at least one alkyl glycoside wherein the alkyl hasfrom 1 to 30 carbon atoms.

Also provided is a method of treating conditions associated with insulinresistance including and not limited to obesity, the metabolic syndrome,type 2 diabetes, hypertension, atherosclerosis or the like, comprisingadministering a drug composition comprising a peptide covalently linkedto at least one alkyl glycoside and delivered to a vertebrate, whereinthe alkyl has from 1 to 30 carbon atoms, 1 to 20 carbons, or further inthe range of 6 to 16 carbon atoms, or 6 to 18 carbons, and whereincovalent linkage of the alkyl glycoside to the peptide increases thestability, bioavailability and/or duration of action of the drug.

Further provided herein is the use of a peptide product described herein(e.g., a peptide product of Formula I-A, Formula III-A, Formula III-B,or Formula V) for the manufacture of a medicament for treatment of anycondition described above and herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Table 1 in FIG. 1 depicts compounds that were prepared by methodsdescribed herein. The specification provides sequences for SEQ. ID. Nos.1-3 and SEQ. ID. Nos. 318-343. Additionally, Table 1 of FIG. 1 providesSEQ. ID Numbers for compounds EU-A300 to EU-A425 having SEQ. ID. NOs.4-129 respectively, as shown in Table 1 of FIG. 1. Compounds in Table 1of FIG. 1, and their respective SEQ. ID. NOs. shown in Table 1 of FIG. 1are hereby incorporated into the specification as filed.

FIG. 2 Table 2 in FIG. 2 depicts compounds that were prepared by methodsdescribed herein. The specification provides SEQ. ID. Nos. 1-3 and SEQ.ID. Nos. 318-343. Additionally, Table 2 of FIG. 2 provides SEQ. IDNumbers for compounds EU-A426 to EU-599 having SEQ. ID. NOs. 130-317respectively, as shown in Table 2 of FIG. 2. Compounds in Table 2 ofFIG. 2, and their respective SEQ. ID. NOs. shown in Table 2 of FIG. 2are hereby incorporated into the specification as filed.

FIG. 3 FIG. 3 illustrates the x-ray crystal structure (Runge, S., et al.(2008) J Biol Chem 283: 11340-7) of the binding site of theextracellular domain of the GLP-1 receptor and 22A illustrates criticalhydrophobic binding elements of the receptor and the ligand exendin-4(Val¹⁹*, Phe²²*, Trp²⁵*, Leu²⁶*) which are mimicked and replaced by thehydrophobic 1′-alkyl portion of the surfactant on the peptides of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are certain covalently modified peptides and/orproteins with improved pharmaceutical properties. Also provided hereinare methods for use of the covalently modified peptides and/or proteinsfor treatment of disorders related to obesity and the metabolicsyndrome.

In some embodiments, the modified peptides and/or proteins comprise apeptide and/or protein covalently attached to a hydrophilic group, a“head” (e.g., a polyol, (e.g., a saccharide)); the hydrophilic group iscovalently attached to a hydrophobic group, a “tail”, thereby generatinga surfactant. In some embodiments, use of hydrophobic-linked glycosidesurfactant (e.g., alkyl glycoside) moieties for covalent modification ofthe peptides or proteins (e.g., glucagon or GLP-1 related peptides orthe like), prolongs the duration of action of the peptides and/orproteins by multiple mechanisms, including formation of depots of thedrug at the site of administration in the body and binding tohydrophobic carrier proteins. In some embodiments, incorporation ofsteric hindrance into peptide and/or protein structure can preventapproach of proteases to the peptide and/or protein product and therebyprevent proteolysis. In some embodiments, surfactant modification (e.g.,covalent attachment of alkyl glycoside class of surfactants) of peptidesand/or proteins as described herein, increases the transport acrossmucosal barriers. Accordingly, the modifications of the peptides and/orproteins described herein provide desirable benefits including and notlimited to, protection from proteolysis, and slowed movement from thesite of administration, thereby leading to prolonged pharmacokineticbehavior (e.g., prolongation of circulating t_(1/2)) and improvedtransmucosal bioavailability.

In some embodiments, interaction of the improved peptides and/orproteins with their receptors is modified in beneficial ways by thetruncation of the sequence, introduction of constraint, and/or theincorporation of steric hindrance. Described herein are novel alkylglycoside reagents that allow for incorporation of both rigidity andsteric hindrance in the modified peptides and/or proteins. In someembodiments, steric hindrance confers receptor selectivity to themodified peptides and/or proteins described herein. In some embodiments,steric hindrance provides protection from proteolysis.

Proteins and peptides undergo numerous physical and chemical changesthat may affect potency and safety. Among these are aggregation, whichincludes dimerization, trimerization, and the formation of higher-orderaggregates such as amyloids. Aggregation is a key issue underlyingmultiple potentially deleterious effects for peptide and/orprotein-based therapeutics, including loss of efficacy, alteredpharmacokinetics, reduced stability or product shelf life, and inductionof undesirable immunogenicity. Bioavailability and pharmacokinetics of aself-associating peptide can be influenced by aggregate size and theease of disruption of the non-covalent intermolecular interactions atthe subcutaneous site (Maji, S. K., et al. (2008) PLoS Biol 6: e17). Insome instances, peptides can aggregate into subcutaneous depots thatdisassociate with t_(1/2) of 30 or more days. Such slow dissolution canlead to favorable effects such as delivery for one month from a singlesc injection causes such a low blood concentration that the peptideappears inactive in vivo. Thus, in some instances, hydrophobicaggregation precludes a peptide's bioavailability and effectiveness(Clodfelter, D. K., et al. (1998) Pharm Res 15: 254-262). The modifiedpeptide products described herein are surfactant-linked and areoptionally designed to allow for either interference with aggregation,or enhanced aggregation, as desired.

Often naturally occurring oligosaccharides that are covalently attachedto proteins do not have surfactant character. In some embodiments,peptide and/or protein products described herein have a covalentlyattached saccharide and an additional hydrophobic group that conferssurfactant character to the modified peptides, thereby allowing fortunability of bioavailability, immunogenicity, and/or pharmacokineticbehavior of the surfactant-modified peptides.

Proteins and peptides modified with oligosaccharides are described in,for example, Jensen, K. J. and Brask, J. (2005) Biopolymers 80: 747-761,through incorporation of saccharide or oligosaccharide structures usingenzymatic (Gijsen, H. J., et al. (1996) Chem Rev 96: 443-474; Sears, P.and Wong, C. H. (1998) Cell Mol Life Sci 54: 223-252; Guo, Z. and Shao,N. (2005) Med Res Rev 25: 655-678) or chemical approaches (Urge, L., etal. (1992) Biochem Biophys Res Commun 184: 1125-1132; Salvador, L. A.,et al. (1995) Tetrahedron 51: 5643-5656; Kihlberg, J., et al. (1997)Methods Enzymol 289: 221-245; Gregoriadis, G., et al. (2000) Cell MolLife Sci 57: 1964-1969; Chakraborty, T. K., et al. (2005) Glycoconj J22: 83-93; Liu, M., et al. (2005) Carbohydr Res 340: 2111-2122; Payne,R. J., et al. (2007) J Am Chem Soc 129: 13527-13536; Pedersen, S. L., etal. (2010) Chembiochem 11: 366-374). Peptides as well as proteins havebeen modified by glycosylation (Filira, F., et al. (2003) Org BiomolChem 1: 3059-3063); (Negri, L., et al. (1999) J Med Chem 42: 400-404);(Negri, L., et al. (1998) Br J Pharmacol 124: 1516-1522); Rocchi, R., etal. (1987) Int J Pept Protein Res 29: 250-261; Filira, F., et al. (1990)Int J Biol Macromol 12: 41-49; Gobbo, M., et al. (1992) Int J PeptProtein Res 40: 54-61; Urge, L., et al. (1992) Biochem Biophys ResCommun 184: 1125-1132; Djedaini-Pilard, F., et al. (1993) TetrahedronLett 34: 2457-2460; Drouillat, B., et al. (1997) Bioorg Med Chem Lett 7:2247-2250; Lohof, E., et al. (2000) Angew Chem Int Ed Engl 39:2761-2764; Gruner, S. A., et al. (2001) Org Lett 3: 3723-3725; Pean, C.,et al. (2001) Biochim Biophys Acta 1541: 150-160; Filira, F., et al.(2003) Org Biomol Chem 1: 3059-3063; Grotenbreg, G. M., et al. (2004) JOrg Chem 69: 7851-7859; Biondi, L., et al. (2007) J Pept Sci 13:179-189; Koda, Y., et al. (2008) Bioorg Med Chem 16: 6286-6296;Yamamoto, T., et al. (2009) J Med Chem 52: 5164-5175).

However, the aforementioned attempts do not describe an additionalhydrophobic group attached to the peptide-linked oligosaccharide.Accordingly, provided herein are modified peptides and/or proteins thatincorporate a hydrophobic group attached to a saccharide and/oroligosaccharide that is covalently attached to the peptide and/orprotein and that allow for tunability of bioavailability, immunogenicityand pharmacokinetic behavior. Accordingly, also provided herein aresurfactant reagents comprising an oligosaccharide and a hydrophobicgroup, that allow for covalent modification of peptides and/or proteinssuch as, for example, glucagon and/or GLP-1 and/or analogs thereof.

Provided herein is the use of saccharide-based surfactants in covalentlinkage to a peptide for improvement of peptide and/or proteinproperties. In some embodiments, surfactant modification (e.g., covalentattachment of alkyl glycoside class of surfactants) of peptides and/orproteins as described herein, increases the transport across mucosalbarriers. In some embodiments, covalent attachment of a surfactant to apeptide and/or protein product reduces or prevents aggregation of thepeptide and/or protein. In some embodiments, the covalently modifiedpeptides and/or proteins are covalently modified glucagon or GLP-1peptides, or analogs thereof, which are modified to improve theirpharmaceutical and medical properties by covalent modification withalkyl glycoside surfactant moieties. These surfactant-modified analogshave increased steric hindrance that hinder proteolysis, slows uptakeand slows clearance from the body.

In certain instances, the effects of surfactants are beneficial withrespect to the physical properties or performance of pharmaceuticalformulations, but are irritating to the skin and/or other tissues and inparticular are irritating to mucosal membranes such as those found inthe nose, mouth, eye, vagina, rectum, buccal or sublingual areas.Additionally, in some instances, surfactants denature proteins thusdestroying their biological function. Since surfactants exert theireffects above the critical micelle concentration (CMC), surfactants withlow CMC's are desirable so that they may be utilized with effectivenessat low concentrations or in small amounts in pharmaceuticalformulations. Accordingly, in some embodiments, surfactants (e.g., alkylglycosides) suitable for peptide modifications described herein have theCMC's less than about 1 mM in pure water or in aqueous solutions. By wayof example only, certain CMC values for alkyl glycosides in water are:Octyl maltoside 19.5 mM; Decyl maltoside 1.8 mM; Dodecyl-β-D-maltoside0.17 mM; Tridecyl maltoside 0.03 mM; Tetradecyl maltoside 0.01 mM;Sucrose dodecanoate 0.3 mM. It will be appreciated that a suitablesurfactant could have a higher or lower CMC depending on the peptideand/or protein that is modified. As used herein, “Critical MicelleConcentration” or “CMC” is the concentration of an amphiphilic component(alkyl glycoside) in solution at which the formation of micelles(spherical micelles, round rods, lamellar structures etc.) in thesolution is initiated. In certain embodiments, the alkyl glycosidesdodecyl, tridecyl and tetradecyl maltoside or glucoside as well assucrose dodecanoate, tridecanoate, and tetradecanoate are possess lowerCMC's and are suitable for peptide and/or protein modificationsdescribed herein.

Insulin Resistance

The risks associated with prolonged hyperglycemia include an increasedrisk of microvascular complications, sensory neuropathy, myocardialinfarction, stroke, macrovascular mortality, and all-cause mortality.Type 2 diabetes is also linked causally with obesity, an additionalglobal epidemic. At least $232 billion were spent on treatment andprevention of diabetes worldwide in 2007, with three quarters of thatamount spent in industrialized countries on the treatment of long-termcomplications and on general care, such as efforts to prevent micro andmacrovascular complications. In 2007, estimated indirect costs ofdiabetes (disability, lost productivity, and premature death due todiabetes) to the United States economy were $58 billion.

Obesity leads to insulin resistance, a decreased ability of the cells inthe body to react to insulin stimulation through decreased numbers ofinsulin receptors and a decreased coupling of those receptors tocritical intracellular signaling systems. The obese state further leadsto the “metabolic syndrome”, a constellation of diseases (insulinresistance, hypertension, atherosclerosis, et al.) with very largehealthcare consequences. If insulin resistance is diagnosed earlyenough, overt type 2 diabetes can be prevented or delayed, withlifestyle interventions aimed at reducing calorie intake and body fatand through drug treatment to normalize glycemic control. Despitetreatment guidelines recommending early, aggressive intervention, manypatients fail to reach targets for glycemic control. Many factorscontribute to the failure to manage type 2 diabetes successfullyincluding psychosocial and economic influences and shortcomings in theefficacy, convenience and tolerability profiles of availableantidiabetic drugs. The peptide and/or protein products described hereinare designed to overcome these shortcomings.

Incretin Effect

The “incretin effect” is used to describe the phenomenon whereby aglucose load delivered orally produces a much greater insulin secretionthan the same glucose load administered intravenously. This effect ismediated by at least two incretin hormones secreted by intestinalL-cells. Glucose-dependent insulinotropic polypeptide (GIP) andglucagon-like peptide 1 (GLP-1) were identified as incretins and it isthought that healthy individuals may derive up to 70% of their prandialinsulin secretory response from the incretin effect.

Normally the incretin peptides are secreted as needed, in response toingested nutrients, and have a short plasma half-life due to degradationby dipeptidyl peptidase IV (DPP-4) enzyme. In people with type 2diabetes, pancreatic responsiveness to GLP-1 is impaired, but theinsulin-secretory response can be restored with pharmacologic doses ofhuman GLP-1 (Kieffer, T. J., et al. (1995) Endocrinology 136:3585-3596). In addition, GLP-1 promotes beta-cell neogenesis andpreservation (Aaboe, K., et al. (2008) Diabetes Obes Metab 10:994-1003). GLP-1 has additional beneficial effects such as on cardiacfunction: for example it improves left ventricular function (Sokos, G.G., et al. (2006) J Card Fail 12: 694-699) in human subjects. GLP-1 alsoslows gastric emptying in humans and reduces appetite (Toft-Nielsen, M.B., et al. (1999) Diabetes Care 22: 1137-1143).

Treatment of diabetes patients with metabolically stable and long-actinganalogs of GLP-1 is described in, for example, Drab, S. R. (2010)Pharmacotherapy 30: 609-624, suffers from issues related to convenienceof use and side effects such as nausea, risk of pancreatitis and thyroidcarcinoma. GLP-1 analogs provide glucose-dependent stimulation ofinsulin secretion and lead to a reduced risk of hypoglycemia. Inaddition, while a number of the current treatments for diabetes causeweight gain, as described below, GLP-1 analogs induce satiety and a mildweight loss. Accordingly, in some embodiments, provided herein are GLP-1analogs that are long acting and are administered at low doses therebyreducing side-effects associated with current treatments.

A number of peptide gut hormones are known to modulate appetite (Sanger,G. J. and Lee, K. (2008) Nat Rev Drug Discov 7: 241-254). Severalpeptides are derived from tissue-specific, enzymatic processing(prohormone convertases; PCs) of the preproglucagon gene product: e.g.glucagon, GLP-1, glucagon-like peptide-2 (GLP-2), glicentin andoxyntomodulin (OXM) (Drucker, D. J. (2005) Nat Clin Pract EndocrinolMetab 1: 22-31; Sinclair, E. M. and Drucker, D. J. (2005) Physiology(Bethesda) 20: 357-365). GLP-1, GLP-2, glicentin and OXM are co-secretedfrom L-cells in the gut in response to feeding. Preproglucagon isalternatively processed (PC2) to produce glucagon in the alpha cells inthe pancreatic islets. The structure of OXM is essentially glucagon witha C-terminal extension of 8 residues.

In addition to the stimulation of insulin biosynthesis and ofglucose-dependent insulin secretion, GLP-1 and its stable mimetics (e.g.Byetta) also cause modest weight loss in animal models (Mack, C. M., etal. (2006) Int J Obes (Lond) 30: 1332-1340) and in Type 2 diabeticpatients (DeFronzo, R. A., et al. (2005) Diabetes Care 28: 1092-1100;Buse, J. B., et al. (2010) Diabetes Care 33: 1255-1261). Glucagoninfusion reduces food intake in man (Geary, N., et al. (1992) Am JPhysiol 262: R975-980), while continuous glucagon treatment of adiposetissue also promotes lipolysis (Heckemeyer, C. M., et al. (1983)Endocrinology 113: 270-276) and weight loss (Salter, J. M., et al.(1960) Metabolism 9: 753-768; Chan, E. K., et al. (1984) Exp Mol Pathol40: 320-327). Glucagon has wide-ranging effects on energy metabolism(Heppner, K. M., et al. (2010) Physiol Behav)). Glucagon, or analogs,can be used in a diagnostic mode for temporary paralysis of theintestinal tract. Thus at least two of the products from PC processingof the preproglucagon protein are linked to satiety and metaboliceffects.

In rodents, repeated intraperitoneal administration of OXM, a thirdproduct of preproglucagon, has been associated with reduced whiteadipose tissue and a reduction in weight compared with controls (Dakin,C. L., et al. (2004) Endocrinology 145: 2687-2695). Oxm reduced foodintake by 19.3% during an intravenous infusion administration tonormal-weight humans and this effect continues for more than 12 hr.after infusion (Cohen, M. A., et al. (2003) J Clin Endocrinol Metab 88:4696-4701). Treatment of volunteers over a 4 week period resulted in asustained satiety effect and weight loss reflecting a decrease in bodyfat (Wynne, K., et al. (2005) Diabetes 54: 2390-2395).

OXM is structurally homologous with GLP-1 and glucagon, and activatesboth the glucagon receptor (GCGR) and the GLP-1 receptor (GLP1R), butwith 10 to 100 fold less potency than the eponymous ligands. Inaddition, study of OXM interactions with GLP1R suggest it might havedifferent effects on beta-arrestin recruitment compared to GLP-1(Jorgensen, R., et al. (2007) J Pharmacol Exp Ther 322: 148-154), thusacting as a “biased” ligand. A unique receptor for OXM was sought for anumber of years, but has not yet been elucidated and it is assumed toact through the GLP1R and GCGR pathways. Accordingly, provided hereinare methods for surfactant modification of gut peptides that allow forinduction of satiety, weight loss, alleviation of insulin resistanceand/or delay in progression of prediabetes to diabetes.

GLP-1

In view of the complex and interacting behavior of the products of thepreproglucagon protein on satiety and metabolism described above,workers from multiple groups have studied the structure activityrelationships on GLP-1 and glucagon structure. Residues throughout thesequences were shown to accept replacement. For example, replacement byAla is well accepted in the N-terminal region of GLP-1, especially at 2,3, 5, 8, 11, and 12 (Adelhorst, K., et al. (1994) J Biol Chem 269:6275-6278).

It was shown that chimeric analogs with the ability to bind to GLP1R andGLCR could be achieved by grafting C-terminal residues from GLP-1 ontothe N-terminus of glucagon (Hjorth, S. A., et al. (1994) J Biol Chem269: 30121-30124). The residue at position 3 (acidic Glu in GLP1 orneutral Gln in Glucagon or OXM) reduces the affinity of glucagon (Runge,S., et al. (2003) J Biol Chem 278: 28005-28010) or OXM (Pocai, A., etal. (2009) Diabetes 58: 2258-2266) for the GlP1R. The effect onmetabolic profile of animals treated with stabilized analogs of GLP-1 orglucagon or OXM with Gln in position 3 was studied (Day, J. W., et al.(2009) Nat Chem Biol 5: 749-757; Druce, M. R., et al. (2009)Endocrinology 150: 1712-1722; Pocai, A., et al. (2009) Diabetes 58:2258-2266). These analogs were designed to have agonistic action on bothGLP1R and on GCGR (Day, J. W., et al. US 2010/0190701 A1).

Chimeric analogs should have the desirable effects of the parenthormones acting on their receptors, and therefore similar to the effectsof OXM, which apparently acts on both GLP-1R and GLCR: glucose-dependentinsulin secretion and satiety, coupled with lipolysis and increasedburning of fat due to glucagon. The analogs were shown to cause thedesired effects of decreased weight and increased burning of fat. Such aprofile would be attractive in the treatment of obesity, but a majorchallenge in obesity treatment is compliance. Although currently knownfill length analogs of glucagon and OXM, respectively, with affinity forboth GLP-1R and GLCR can result in weight loss, these analogs are notoptimized for the high bioavailability, pharmaceutical properties, andconvenient delivery to patients that are necessary for optimal drugtreatment regimens. Accordingly, provided herein are analogs of gutpeptides (e.g., GLP, OXM, glucagon or the like) that allow for highbioavailability and/or long lasting effects for improved therapeuticoutcome in treatment of conditions such as obesity and/or diabetesand/or the metabolic syndrome.

Additional factors for optimized treatment of the metabolic syndrome anddiabetes with OXM-like molecules relate to the duration of treatment andthe amount of glucagon action. For example, continuous treatment withanalogs that activate GLP-1 and glucagon receptors (the OXMpharmacological profile) can result in very large and rapid loss of fatmass (Day, J. W., et al. (2009) Nat Chem Biol 5: 749-757), but it canalso cause the loss of lean muscle mass (Kosinski, J. R., et al. (2012)Obesity (Silver Spring): doi: 10.1038/oby.2012.67), which is unfavorablefor a pharmaceutical in this class. For example, in the research articleby Kosinski, J. R., et al., the natural hormone Oxm is administeredcontinuously for 14 days from an Alzet minipump and results in adecrease of 30% in fat mass, but also caused a 7% decrease in lean mass(muscle).

Glucagon action is known to stimulate glycogenolysis, lipolysis and theincreased burning of fat, but can also have catabolic effects on muscle.A successful treatment using an agent that combines GLP-1 and glucagonaction (the OXM profile) will need to optimally cause the satiety andpotentiated glucose-dependent insulin secretion of a GLP-1 analog with ajudicious amount of glucagon action (fat burning). In addition,intermittent use of such an agent will provide the desired clinicalprofile of moderate, continuous weight loss, through loss of fat mass,with minimized loss of lean mass. Provided herein are molecules with adesirable combination of GLP-1 and OXM action as well as a tunablepharmacokinetic/pharmacodynamic profile to allow optimum use in therapy(for example in the metabolic syndrome, diabetes, obesity, and thelike).

In one embodiment, the compounds of Formula I-A, III-A, III-B andFormula V are designed to provide either glucagon-like activity or GLP-1like activity. In a further embodiment, the compounds of Formula I-A,III-A, III-B and Formula V provide tunable activity. For example, in oneinstance, the peptide products described herein (e.g., compounds inTable 1 of FIG. 1 and Table 2 of FIG. 2) have an EC50 of less than about500 nM, preferably less than about 50 nM, more preferably less thanabout 20 nM at receptors for both glucagon, and GLP-1. In anotherinstance, the peptide products described herein (e.g., compounds inTable 1 of FIG. 1 and Table 2 of FIG. 2) are more potent (e.g., EC50 ofless than 10 nM, preferably less than 5 nM, more preferably about 1 nM)for the GLP-1 receptor and less potent for the glucagon receptor (e.g.,EC50 of less than 50 nM, preferably less than about 20 nM, morepreferably about 5 nM) for the glucagon receptor. This tunability ofbiological activity allows for some retention of a judicious amount ofglucagon action, thereby allowing for fat burning to occur, while alsoretaining the beneficial effects of potentiated glucose-dependentinsulin secretion. OXM is structurally homologous with GLP-1 andglucagon, and activates both the glucagon receptor (GCGR) and the GLP-1receptor (GLP1R). Accordingly, in some embodiments, the compounds ofFormula I-A, Formula III-A, Formula III-B and Formula V provide atunable OXM-like biological activity. In some specific embodiments, thepeptide products described herein comprise a peptide having amino acidresidues 1-17 of GLP-1 and/or analogs thereof (e.g., analogs comprisingmodified non-natural amino acid replacements as described herein,cyclized lactam linkages as described herein, surfactant modificationsas described herein, or a combination thereof). In some otherembodiments, the peptide products described herein comprise a peptidehaving amino acid residues 1-16 of GLP-1 and/or analogs thereof (e.g.,analogs comprising modified non-natural amino acid replacements asdescribed herein, cyclized lactam linkages as described herein,surfactant modifications as described herein, or a combination thereof).In additional embodiments, the peptide products described hereincomprise a peptide having amino acid residues 1-18 of GLP-1 and/oranalogs thereof (e.g., analogs comprising modified non-natural aminoacid replacements as described herein, cyclized lactam linkages asdescribed herein, surfactant modifications as described herein, or acombination thereof). Additionally the peptide products described hereincomprise one or more residues (e.g., Aib, Ac4C) which provide helixstabilization of the designed compounds of Formula I-A, Formula III-A,Formula III-B, Formula V, and compounds in Table 1 of FIG. 1, and Table2 of FIG. 2.

It is believed that the glucagon subfamily of ligands bind to theirreceptors in a two domain mode common to a number of the class B ofreceptors (secretin class, G Protein-coupled Receptors (GPCR)). ForGLP-1 it is felt that there is a N-terminal region of from residue 1 toabout residue 16 which binds to the tops of the transmembrane helicies(juxtomembrane region) and a helical C-terminal region from 17 to 31which binds to the large, extracellular, N-terminal extension (ECD) ofthe receptor. The binding of these ligands focuses on the fact thatN-terminally truncated analogs of these peptide ligands can still retainsubstantial binding affinity and selectivity for just the isolated ECDregion of the receptor. Therefore it has been suggested that theN-terminal region is responsible for receptor activation while theC-terminal region is responsible for binding. It recently has been shownthat short, N-terminal analogs of GLP-1 can be both potent binders aswell as receptor activators (Mapelli, C., et al. (2009) J Med Chem 52:7788-7799; Haque, T. S., et al. (2010) Peptides 31: 950-955; Haque, T.S., et al. (2010) Peptides 31: 1353-1360).

In addition, study of an x-ray crystal structure (Runge, S., et al.(2008) J Biol Chem 283: 11340-7) of the N-terminal region of the GLP1Rwith a truncated antagonist analogs of the GLP-1 mimic, exendin-4(Byetta), bound in this region show that a critical ligand-bindingregion in the ECD is of high hydrophobicity (FIG. 3). The sequence ofexendin-4 beyond Glu 15 interacts as an amphiphilic helix with this veryhydrophobic region (Val¹⁹*, Phe²²*, Trp²⁵*, Leu²⁶*). In one embodiment,truncated N-terminal fragments of GLP-1 or glucagon are modified to bindto GLCR and are covalently linked to a surfactant. The hydrophobic1′-alkyl portion of the surfactant mimics and replaces the C-terminalregion of the native hormone ligand and increases the peptides potency,efficacy, and duration of action. In addition, such analogs have majoradvantages due to their smaller size, which reduces their complexity,synthesis costs, and susceptibility to proteolysis. In addition smallerpeptides are more readily absorbed through the nasal mucosa or gutenterocyte barrier.

Hypoglycemia is a condition of low blood sugar that can belife-threatening and is increasingly seen as more aggressive treatmentof elevated blood sugar by intensive insulin treatment is being used inmore patients. Hypoglycemia is seen when blood glucose levels drop toolow to provide enough energy to the brain and muscles for the body'sactivities. Glucagon can be used to treat this condition and does so bystimulating the liver to break down glycogen to generate glucose andcause the blood glucose levels to rise toward the normal value. Analogsof glucagon that retain the ability to activate the GLCR may be used toachieve this desirable effect on blood glucose levels.

Analogs of GLP-1 that activate the GLP1R stimulate the production and,in the presence of elevated blood glucose levels, release of insulinfrom the pancreas. This action results in efficient control andnormalization of blood glucose levels, as seen with current productssuch as exenatide (Byetta®). In addition, such products appear toproduce a decreased appetite and slow the movement of food from thestomach. Thus they are effective in treatment of diabetes throughmultiple mechanisms. Analogs that combine the effects of glucagon andGLP-1 that activate both the GLCR and the GLP1R may offer a benefit inthe treatment of diabetes through a concerted action to suppressappetite, release insulin in a glucose-dependent fashion, assist in theprotection from hypoglycemia and accelerate the burning of fat.

Such methods for treating hyperglycemia, including diabetes, diabetesmellitus type I, diabetes mellitus type II, or gestational diabetes,either insulin-dependent or non-insulin dependent, are expected to beuseful in reducing complications of diabetes including nephropathy,retinopathy and vascular disease. Applications in cardiovascular diseaseencompass microvascular as well as macrovascular disease (Davidson, M.H., (2011) Am J Cardiol 108[suppl]:33B-41B; Gejl, M., et al. (2012) JClin Endocrinol Metab 97:doi:10.1210/jc.2011-3456), and includetreatment for myocardial infarction. Such methods for reducing appetiteor promoting loss of body weight are expected to be useful in reducingbody weight, preventing weight gain, or treating obesity of variouscauses, including drug-induced obesity, and reducing complicationsassociated with obesity including vascular disease (coronary arterydisease, stroke, peripheral vascular disease, ischemia reperfusion,etc.), hypertension, onset of diabetes type II, hyperlipidemia andmusculoskeletal diseases.

As used herein, the term glucagon or GLP-1 analogs includes allpharmaceutically acceptable salts or esters thereof.

Peptides and Analogs Thereof

In one aspect, the peptides that are covalently modified and aresuitable for methods described herein are truncated analogs of glucagonand/or the related hormone GLP-1, including and not limited to:

Glucagon:

(SEQ. ID. NO. 331) His₁-Ser₂-Gln₃-Gly₄-Thr₅ Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Arg₁₈-Ala₁₉-Gln₂₀-Asp₂₁-Phe₂₂-Val₂₃-Gln₂₄-Trp₂₅-Leu₂₆-Met₂₇-Asn₂₈- Thr₂₉

Oxyntomodulin:

(SEQ. ID. NO. 332) His₁-Ser₂-Gln₃-Gly₄-Thr₅ Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Arg₁₈-Ala₁₉-Gln₂₀-Asp₂₁-Phe₂₂-Val₂₃-Gln₂₄-Trp₂₅-Leu₂₆-Met₂₇-Asn₂₈-Thr₂₉-Lys₃₀-Arg₃₁-Asn₃₂-Arg₃₃-Asn₃₄-Asn₃₅-Ile₃₆-Ala₃₇

GLP-1 (Using Glucagon Numbering):

(SEQ. ID. NO. 333) His₁-A1a₂-Glu₃-Gly₄-Thr₅ Phe₆-Thr₇-Ser₈-Asp₉-Val₁₀-Ser₁₁-Ser₁₂-Tyr₁₃-Leu₁₄-Glu₁₅-Gly₁₆-Gln₁₇-Ala₁₈-Ala₁₉-Lys₂₀-Glu₂₁-Phe₂₂-Ile₂₃-Ala₂₄-Trp₂₅-Leu₂₆- Val₂₇-Lys₂₈-Gly₂₉-Arg₃₀

In some embodiments, a peptide product described herein has thestructure of Formula V:

FORMULA V (SEQ. ID. NO. 334)aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-aa₃₀-aa₃₁-aa₃₂-aa₃₃-aa₃₄-aa₃₅- aa₃₆-aa₃₇-Zwherein:

-   -   U is a linking amino acid;    -   X is a surfactant-linked to the side chain of U;    -   Z is OH, or —NH—R³, wherein R³ is H or C₁-C₁₂ substituted or        unsubstituted alkyl;    -   aa₁ is His, N—Ac-His, pGlu-His or N—R³-His;    -   aa₂ is Ser, Ala, Gly, Aib, Ac4c or Ac5c;    -   aa₃ is Gln, or Cit;    -   aa₄ is Gly, or D-Ala;    -   aa₅ is Thr, or Ser,    -   aa₆ is Phe, Trp, F2Phe, Me2Phe, or Nal(2);    -   aa₇ is Thr, or Ser,    -   aa₈ is Ser, or Asp;    -   aa₉ is Asp, or Glu;    -   aa₁₀ is Tyr, Leu, Met, Nal(2), Bip, or Bip2EtMeO;    -   aa₁₁ is Ser, Asn, or U(X);    -   aa₁₂ is Lys, Glu, Ser, Arg, or U(X);    -   aa₁₃ is absent, Tyr, Gln, Cit, or U(X);    -   aa₁₄ is absent, Leu, Met, Nle, or U(X);    -   aa₁₅ is absent, Asp, Glu, or U(X);    -   aa₁₆ is absent, Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or U(X);    -   aa₁₇ is absent, Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c, or        U(X);    -   aa₁₈ is absent, Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X);    -   aa₁₉ is absent, Ala, Val, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₀ is absent, Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c, or        U(X);    -   aa₂₁ is absent, Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₂ is absent, Phe, Trp, Nal(2), Aib, Ac4c, Ac5c, or U(X);    -   aa₂₃ is absent, Val, Ile, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₄ is absent, Gln, Ala, Glu, Cit, or U(X);    -   aa₂₅ is absent, Trp, Nal(2), or U(X);    -   aa₂₆ is absent, Leu, U(X);    -   aa₂₇ is absent, Met, Val, Nle, Lys, or U(X);    -   aa₂₈ is absent, Asn, Lys, or U(X);    -   aa₂₉ is absent, Thr, Gly, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₀ is absent, Lys, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₁ is absent, Arg, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₂ is absent, Asn, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₃ is absent, Arg, Aib, Ac5c, or U(X);    -   aa₃₄ is absent, Asn, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₅ is absent, Asn, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₆ is absent, lie, Aib, Ac4c, Ac5C, or U(X);    -   aa₃₆ is absent, Ala, Aib, Ac4c, Ac5C, or U(X);    -   aa₃₇ absent or U(X);

provided that one, or at least one of aa₁₁-aa₃₇ is U(X).

In specific embodiments, the linking amino acid U, is a diamino acidlike Lys or Orn, X is a modified surfactant from the 1-alkyl glycosideclass linked to U, and Z is OH, or —NH—R₂, wherein R³ is H or C₁-C₁₂; ora PEG chain of less than 10 Da.

In some embodiments, a peptide product described herein has thestructure of Formula III-B:

FORMULA III-B (SEQ. ID. NO. 3)His₁-aa₂-aa₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂- aa₂₃-Z

-   -   wherein:        -   Z is OH, or —NH—R³, wherein R³ is H or substituted or            unsubstituted C₁-C₁₂ alkyl; or a        -   PEG chain of less than 10 Da;            -   aa₂ is Ser, Ala, Gly, Aib, Ac4c, or Ac5c;            -   aa₃ is Gln, or Cit;            -   aa₆ is Phe, Trp, F2Phe, Me2Phe, MePhe, or Nal2;            -   aa₁₀ is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO;            -   aa₁₁ is Ser, Asn, or U.            -   aa₁₂ is is Lys, Glu, Ser or U(X);            -   aa₁₃ is absent or Tyr, Gln, Cit, or U(X);            -   aa₁₄ is absent or Leu, Met, Nle, or U(X);            -   aa₁₅ is absent or Asp, Glu, or U(X);            -   aa₁₆ is absent or Ser, Gly, Glu, Aib, Ac4c, Ac5c, Lys,                R, or U(X);            -   aa₁₇ is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c,                Ac5c, or U(X);            -   aa₁₈ is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or                U(X);            -   aa₁₉ is absent or Ala, Val, Aib, Ac4c, Ac5c, or U(X);            -   aa₂₀ is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c,                Ac5c, or U(X);            -   aa₂₁ is absent or Asp, Glu, Leu, Aib, Ac4c, Ac5c, or                U(X);            -   aa₂₂ is absent or Phe, Aib, Ac4c, Ac5c, or U(X)            -   aa₂₃ is absent or Val, Ile, Aib, Ac4c, Ac5c, or U(X);        -   wherein any two of aa₁-aa₂ are optionally cyclized through            their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₆, aa₁₇, aa₁₈, aa₁₉,        aa₂₀, aa₂₁, aa₂₂, aa₂₃ or aa₂₄ is the natural or unnatural amino        acid U covalently attached to X.

In some specific embodiments of Formula III-A, Formula III-B and FormulaV, X has the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C—O)—NH—;        -   W² is —O—; and        -   R² is a bond

In some of the embodiments described above, R^(1a) is a C₁-C₂₀ alkylgroup, a C₈-C₂₀ alkyl group, C₁₂-18 alkyl group or C₁₄-C₁₈ alkyl group.

In some embodiments of Formula III-B, U is any linker amino aciddescribed herein.

Table 1 in FIG. 1 and Table 2 in FIG. 2 illustrate certain examples ofpeptides that covalently linked with surfactants as described herein.

Contemplated within the scope of embodiments presented herein arepeptide products of Formula I-A, Formula III-A, Formula III-B or FormulaV, wherein the peptide product comprises one, or, more than onesurfactant groups (e.g., group X having the structure of Formula I). Inone embodiment, a peptide product of Formula I-A, Formula III-A, FormulaIII-B or Formula V, comprises one surfactant group. In anotherembodiment, a peptide product of Formula I-A, Formula III-A, FormulaIII-B or Formula V, comprises two surfactant groups. In yet anotherembodiment, a peptide product of Formula I-A, Formula III-A, FormulaIII-B or Formula V, comprises three surfactant groups.

Recognized herein is the importance of certain portions of SEQ. ID. NO.331 for the treatment of conditions associated with insulin resistanceand/or cardiovascular conditions. Accordingly, provided herein is amethod of treating diabetes in an individual in need thereof comprisingadministration of a therapeutically effective amount of a glucagonanalog comprising amino acid residues aa₁-aa₁₇ of SEQ. ID. NO. 331 tothe individual in need thereof.

In a further embodiment, provided herein is a method of treatingdiabetes in an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₈ of SEQ. ID. NO. 331 to the individual in needthereof.

In another embodiment, provided herein is a method of treating diabetesin an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₉ of SEQ. ID. NO. 331 to the individual in needthereof.

In another embodiment, provided herein is a method of treating diabetesin an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₂₀ of SEQ. ID. NO. 331 to the individual in needthereof.

In an additional embodiment, the administration of the said glucagonanalog described above causes weight loss.

Recognized herein is the importance of certain portions of SEQ. ID. NO.1 for the treatment of conditions associated with insulin resistanceand/or cardiovascular conditions. Accordingly, provided herein is amethod of treating diabetes in an individual in need thereof comprisingadministration of a therapeutically effective amount of a glucagonanalog comprising amino acid residues aa₁-aa₁₇ of SEQ. ID. NO. 1 to theindividual in need thereof.

In a further embodiment, provided herein is a method of treatingdiabetes in an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₈ of SEQ. ID. NO. 1 to the individual in needthereof.

In another embodiment, provided herein is a method of treating diabetesin an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₉ of SEQ. ID. NO. 1 to the individual in needthereof.

In another embodiment, provided herein is a method of treating diabetesin an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₂₀ of SEQ. ID. NO. 1 to the individual in needthereof.

In an additional embodiment, the administration of the said glucagonanalog described above causes weight loss.

In any of the embodiments described above, the said glucagon analog ismodified with a surfactant X of Formula I:

-   -   wherein:        -   R^(1a) is independently, at each occurrence, a bond, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, a substituted            or unsubstituted aralkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, a bond, H, a substituted or unsubstituted C₁-C₃₀            alkyl group, a substituted or unsubstituted alkoxyaryl            group, or a substituted or unsubstituted aralkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O—, —CH₂— or —S—;        -   R² is independently, at each occurrence, a bond to U, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, or a            substituted or unsubstituted aralkyl group, —NH₂, —SH,            C₂-C₄-alkene, C₂-C₄-alkync, —NH(C—O)—CH₂—Br,            —(CH₂)_(m)-maleimide, or —N₃;        -   n is 1, 2 or 3; and        -   m is 1-10.

In a specific embodiment, the said glucagon analog is modified with asurfactant, X having the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond

In some of the embodiments described above, R^(1a) is a C₁-C₂₀ alkylgroup, a C₈-C₂₀ alkyl group, C₁₂-C₁₈ alkyl group or C₁₄-C₁₈ alkyl group.

As used herein, the term diabetes includes both Type 1 and Type 2diabetes. Accordingly, in some embodiments the methods described hereincomprise administration of any compound described herein includingcompounds of Formula II, III-A, III-B and/or Formula V, and/or compoundsdescribed in Table 1 of FIG. 1 and Table 2 of FIG. 2 to an individualsuffering from Type 1 diabetes. In some other embodiments, the methodsdescribed herein comprise administration of any compound describedherein including compounds of Formula II, III-A, III-B and/or Formula V,and/or compounds described in Table 1 of FIG. 1 and Table 2 of FIG. 2 toan individual suffering from Type 2 diabetes.

Also provided herein is a method of treating a cardiovascular disease inan individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₇ of SEQ. ID. NO. 331 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disease inan individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₈ of SEQ. ID. NO. 331 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disease inan individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₉ of SEQ. ID. NO. 331 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disease inan individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₂₀ of SEQ. ID. NO. 331 to the individual in needthereof.

In some cases for the embodiments described above, the said glucagonanalog is administered when the cardiovascular disease is associatedwith an ischemic event.

Also provided herein is a method of treating a cardiovascular disease inan individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₇ of SEQ. ID. NO. 1 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disease inan individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₈ of SEQ. ID. NO. 1 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disease inan individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₉ of SEQ. ID. NO. 1 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disease inan individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₂₀ of SEQ. ID. NO. 1 to the individual in needthereof.

In some cases for the embodiments described above, the said glucagonanalog is administered when the cardiovascular disease is associatedwith an ischemic event.

In any of the embodiments described above, the said glucagon analog ismodified with a surfactant X of Formula I:

-   -   wherein:        -   R^(1a) is independently, at each occurrence, a bond, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, a substituted            or unsubstituted aralkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, a bond, H, a substituted or unsubstituted C₁-C₃₀            alkyl group, a substituted or unsubstituted alkoxyaryl            group, or a substituted or unsubstituted aralkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C—O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O—, —CH₂— or —S—;        -   R² is independently, at each occurrence, a bond to U, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, or a            substituted or unsubstituted aralkyl group, —NH₂, —SH,            C₂-C₄-alkene, C₂-C₄-alkyne, —NH(C═O)—CH₂—Br,            —(CH₂)_(m)-maleimide, or —N₃;        -   n is 1, 2 or 3; and        -   m is 1-10.

In a specific embodiment, the said glucagon analog is modified with asurfactant, X having the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W is —O—; and        -   R² is a bond.

In some of the embodiments described above, R^(1a) is a C₁-C₂₀ alkylgroup, a C₈-C₂₀ alkyl group, C₁₂-C₁₈ alkyl group or C₁₄-C₁₈ alkyl group.

Modifications at the amino or carboxyl terminus may optionally beintroduced into the peptides (e.g., glucagon or GLP-1) (Nestor, J. J.,Jr. (2009) Current Medicinal Chemistry 16: 4399-4418). For example, thepeptides can be truncated or acylated on the N-terminus to yieldpeptides analogs exhibiting low efficacy, partial agonist and antagonistactivity, as has been seen for some peptides (Gourlet, P., et al. (1998)Eur J Pharmacol 354: 105-111, Gozes, I. and Furman, S. (2003) Curr PharmDes 9: 483-494), the contents of which is incorporated herein byreference). For example, deletion of the first 6 residues of bPTH yieldsantagonistic analogs (Mahaffey, J. E., et al. (1979) J Biol Chem 254:6496-6498; Goldman, M. E., et al. (1988) Endocrinology 123: 2597-2599)and a similar operation on peptides described herein generates potentantagonistic analogs. Other modifications to the N-terminus of peptides,such as deletions or incorporation of D-amino acids such as D-Phe alsocan give potent and long acting agonists or antagonists when substitutedwith the modifications described herein such as long chain alkylglycosides. Such agonists and antagonists also have commercial utilityand are within the scope of contemplated embodiments described herein.

Also contemplated within the scope of embodiments described herein aresurfactants covalently attached to peptide analogs, wherein the nativepeptide is modified by acetylation, acylation, PEGylation,ADP-ribosylation, amidation, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-link formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, (Nestor, J. J., Jr. (2007) Comprehensive Medicinal ChemistryII 2: 573-601, Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:4399-4418, Creighton, T. E. (1993, Wold, F. (1983) PosttranslationalCovalent Modification of Proteins 1-12, Seifter, S. and Englard, S.(1990) Methods Enzymol 182: 626-646, Rattan, S. I., et al. (1992) Ann NY Acad Sci 663: 48-62). Also contemplated within the scope ofembodiments described herein are peptides that are branched or cyclic,with or without branching. Cyclic, branched and branched circularpeptides result from post-translational natural processes and are alsomade by suitable synthetic methods. In some embodiments, any peptideproduct described herein comprises a peptide analog described above thatis then covalently attached to an alkyl-glycoside surfactant moiety.

Also contemplated within the scope of embodiments presented herein arepeptide chains substituted in a suitable position by the substitution ofthe analogs claimed herein by acylation on a linker amino acid, at forexample the ε-position of Lys, with fatty acids such as octanoic,decanoic, dodecanoic, tetradecanoic, hexadecanoic, octadecanoic,3-phenylpropanoic acids and the like, with saturated or unsaturatedalkyl chains (Zhang, L. and Bulaj, G. (2012) Curr Med Chem 19:1602-1618). Non-limiting, illustrative examples of such analogs are:

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Lys(N-epsilon-dodecanoyl)₁₈-Aib₁₉-NH₂,(SEQ. ID. NO. 335)

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Lys(N-epsilon-tetradecanoyl)₁₈-Ac4c₁₉-NH₂,(SEQ. ID. NO. 336)

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Lys(N-epsilon-hexadecanoyl)₁₈-Aib₁₉-NH₂,(SEQ. ID. NO. 337)

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-Arg₁₇-Lys(N-epsilon-dodecanoyl)₁₈-NH₂,(SEQ. ID. NO. 338)

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-Arg₁₇-Lys(N-epsilon-tetradecanoyl)₁₈-NH₂,(SEQ. ID. NO. 339)

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-Arg₁₇-Lys(N-epsilon-hexadecanoyl)₁₈-NH₂,(SEQ. ID. NO. 340)

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Lys(N-epsilon-(gamma-glutamyl)-N-alpha-tetradecanoyl))₁₈-Aib₁₉-NH₂,(SEQ. ID. NO. 341) and the like.

In further embodiments, a peptide chain is optionally substituted in asuitable position by reaction on a linker amino acid, for example thesulfhydryl of Cys, with a spacer and a hydrophobic moiety such as asteroid nucleus, for example a cholesterol moiety. In some of suchembodiments, the modified peptide further comprises one or more PEGchains. Non-limiting examples of such molecules are:

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-Arg₁₇-Cys(S-(3-(PEG4-aminoethylacetamide-Cholesterol)))₁₈-Aib₁₉-NH₂,(SEQ. ID. NO. 342)

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser-Asp₉-Tyr₁₀-Ser₁₁-cyclo(Glu₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Lys₁₆)-Arg₁₇-Cys(S-(3-(PEG4-aminoethylacetamide-Cholesterol)))₁₈-NH₂.(SEQ. ID. NO. 343)

Aside from the twenty standard amino acids, there are a vast number of“nonstandard amino acids” or unnatural amino acids that are known to theart and that may be incorporated in the compounds described herein, asdescribed above. Other nonstandard amino acids are modified withreactive side chains for conjugation (Gauthier, M. A. and Klok, H. A.(2008) Chem Commun (Camb) 2591-2611; de Graaf, A. J., et al. (2009)Bioconjug Chem 20: 1281-1295). In one approach, an evolved tRNA/tRNAsynthetase pair and is coded in the expression plasmid by the ambersuppressor codon (Deiters, A, et al. (2004). Bio-org. Med. Chem. Lett.14, 5743-5). For example, p-azidophenylalanine was incorporated intopeptides and then reacted with a functionalized surfactant, or a PEGpolymer having an acetylene moiety in the presence of a reducing agentand copper ions to facilitate an organic reaction known as “Huisgen[3+2] cycloaddition.” A similar reaction sequence using the reagentsdescribed herein containing an acetylene modified alkyl glycoside or PEGmodified glycoside will result in PEGylated or alkyl glycoside modifiedpeptides. For peptides of less than about 50 residues, standard solidphase synthesis is used for incorporation of said reactive amino acidresidues at the desired position in the chain. Such surfactant-modifiedpeptides and/or proteins offer a different spectrum of pharmacologicaland medicinal properties than peptides modified by PEG incorporationalone.

The skilled artisan will appreciate that numerous permutations of thepeptide analogs are possible and, provided that an amino acid sequencehas an incorporated surfactant moiety, will possess the desirableattributes of surfactant modified peptide products described herein.

Certain Definitions

As used in the specification, “a” or “an” means one or more. As used inthe claim(s), when used in conjunction with the word “comprising,” thewords “a” or “an” mean one or more. As used herein, “another” means atleast a second or more.

As used herein, the one- and three-letter abbreviations for the variouscommon amino acids are as recommended in Pure Appl. Chem. 31, 639-645(1972) and 40, 277-290 (1974) and comply with 37 CFR § 1.822 (55 FR18245, May 1, 1990). The abbreviations represent L-amino acids unlessotherwise designated as D- or DL. Certain amino acids, both natural andnon-natural, are achiral, e.g., glycine, Ca-diethylglycine (Deg),α-amino-isobutyric acid (Aib), 1-aminocyclobutane-1-carboxylic acid(Ac4c), 1-aminocyclopentane-1-carboxylic acid (Ac5c),1-aminocyclohexane-1-carboxylic acid (Ac6c). Analogs of glutamineinclude citrulline (Cit). All peptide sequences are presented with theN-terminal amino acid on the left and the C-terminal amino acid on theright.

An “alkyl” group refers to an aliphatic hydrocarbon group. Reference toan alkyl group includes “saturated alkyl” and/or “unsaturated alkyl”.The alkyl group, whether saturated or unsaturated, includes branched,straight chain, or cyclic groups. A “substituted” alkyl group issubstituted with one or more additional group(s). In certainembodiments, the one or more additional group(s) are individually andindependently selected from amide, ester, alkyl, cycloalkyl,heteroalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, ester,alkylsulfone, arylsulfone, cyano, halogen, alkoyl, alkoyloxo,isocyanato, thiocyanato, isothiocyanato, nitro, haloalkyl, haloalkoxy,fluoroalkyl, amino, alkyl-amino, dialkyl-amino, amido, oxo, hydrophobicnatural product such as a steroid, an aralkyl chain (includingalkoxyaryl), alkyl chain containing an acyl moiety, or the like. In someembodiments, an alkyl group is linked to the Nα-position of a residue(e.g., Tyr or Dmt) in a peptide. This class is referred to as N-alkyland comprises straight or branched alkyl groups from C₁-C₁₀, or an arylsubstituted alkyl group such as benzyl, phenylethyl and the like. Insome embodiments, an alkyl moiety is a 1-alkyl group that is inglycosidic linkage (typically to the 1-position of, for example,glucose) to the saccharide moiety. Such a 1-alkyl group is a C₁-C₃₀alkyl group.

An “aryl” group refers to an aromatic ring wherein each of the atomsforming the ring is a carbon atom. Aryl rings described herein includerings having five, six, seven, eight, nine, or more than nine carbonatoms. Aryl groups are optionally substituted with substituents selectedfrom halogen, alkyl, acyl, alkoxy, alkylthio, sulfonyl, dialkyl-amino,carboxyl esters, cyano or the like. Examples of aryl groups include, butare not limited to phenyl, and naphthalenyl.

The term “acyl” refers to a C₁-C₂₀ acyl chain. This chain may comprise alinear aliphatic chain, a branched aliphatic chain, a chain containing acyclic alkyl moiety, a hydrophobic natural product such as a steroid, anaralkyl chain, or an alkyl chain containing an acyl moiety.

The term “steroid nucleus” refers to the core of steroids comprising anarrangement of four fused rings designated A, B, C and D as shown below:

Examples of steroid nucleus containing moieties include, and are notlimited to, cholesterol and the like.

As used herein, a “therapeutic composition” can comprise an admixturewith an aqueous or organic carrier or excipient, and can be compounded,for example, with the usual nontoxic, pharmaceutically acceptablecarriers for tablets, pellets, capsules, lyophilizates, suppositories,solutions, emulsions, suspensions, or other form suitable for use. Thecarriers, in addition to those disclosed above, can include alginate,collagen, glucose, lactose, mannose, gum acacia, gelatin, mannitol,starch paste, magnesium trisilicate, talc, corn starch, keratin,colloidal silica, potato starch, urea, medium chain lengthtriglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition, auxiliary stabilizing, thickening or coloring agents can beused, for example a stabilizing dry agent such as triulose.

As used herein, a “pharmaceutically acceptable carrier” or “therapeuticeffective carrier” is aqueous or nonaqueous (solid), for examplealcoholic or oleaginous, or a mixture thereof, and can contain asurfactant, emollient, lubricant, stabilizer, dye, perfume,preservative, acid or base for adjustment of pH, a solvent, emulsifier,gelling agent, moisturizer, stabilizer, wetting agent, time releaseagent, humectant, or other component commonly included in a particularform of pharmaceutical composition. Pharmaceutically acceptable carriersare well known in the art and include, for example, aqueous solutionssuch as water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, and oils such as olive oil orinjectable organic esters. A pharmaceutically acceptable carrier cancontain physiologically acceptable compounds that act, for example, tostabilize or to increase the absorption of specific inhibitor, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients.

As used herein, a “insulin-resensitizing” amount of a peptide product isan amount that increases the body's response to endogenous orexogenously administered insulin, typically while reducing body weight,in an individual in need thereof as evidenced by, for example, an oralglucose challenge test or euglycemic clamp test.

The pharmaceutical compositions can also contain other pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such “substances” include, but are not limited to, pHadjusting and buffering agents, tonicity adjusting agents and the like,for example, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, etc. Additionally, the peptide, or variantthereof, suspension may include lipid-protective agents which protectlipids against free-radical and lipid-peroxidative damages on storage.Lipophilic free-radical quenchers, such as alpha-tocopherol andwater-soluble iron-specific chelators, such as ferrioxamine, aresuitable.

As used herein, a “surfactant” is a surface active agent that modifiesinterfacial tension of water. Typically, surfactants have one lipophilicand one hydrophilic group or region in the molecule. Broadly, the groupincludes soaps, detergents, emulsifiers, dispersing and wetting agents,and several groups of antiseptics. More specifically, surfactantsinclude stearyltriethanolamine, sodium lauryl sulfate, sodiumtaurocholate, laurylaminopropionic acid, lecithin, benzalkoniumchloride, benzethonium chloride and glycerin monostearate; andhydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone,polyethyleneglycol (PEG), carboxymethylcellulose sodium,methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose andhydroxypropylcellulose or alkyl glycosides. In some embodiments, asurfactant is a non-ionic surfactant (e.g., an alkyl glycosidesurfactant). In some embodiments, a surfactant is an ionic surfactant.

As used herein, “alkyl glycoside” refers to any sugar joined by alinkage to any hydrophobic alkyl, as is known in the art. Thehydrophobic alkyl can be chosen of any desired size, depending on thehydrophobicity desired and the hydrophilicity of the saccharide moiety.In one aspect, the range of alkyl chains is from 1 to 30 carbon atoms;or from 6 to 16 carbon atoms.

As used herein, “saccharide” is inclusive of monosaccharides,oligosaccharides or polysaccharides in straight chain or ring forms.Oligosaccharides are saccharides having two or more monosaccharideresidues. Some examples of the many possible saccharides suitable foruse in functionalized form include glucose, galactose, maltose,maltotriose, maltotetraose, sucrose, trehalose or the like.

As used herein, “sucrose esters” are sucrose esters of fatty acids.Sucrose esters can take many forms because of the eight hydroxyl groupsin sucrose available for reaction and the many fatty acid groups, fromacetate on up to larger, more bulky fats that can be reacted withsucrose. This flexibility means that many products and functionalitiescan be tailored, based on the fatty acid moiety used. Sucrose estershave food and non-food uses, especially as surfactants and emulsifiers,with growing applications in pharmaceuticals, cosmetics, detergents andfood additives. They are biodegradable, non-toxic and mild to the skin.

As used herein, a “suitable” alkyl glycoside means one that is nontoxicand nonionic. In some instances, a suitable alkyl glycoside reduces theimmunogenicity or aggregation and increases the bioavailability of acompound when it is administered with the compound via the ocular,nasal, nasolacrimal, sublingual, buccal, inhalation routes or byinjection routes such as the subcutaneous, intramuscular, or intravenousroutes.

A “linker amino acid” is any natural or unnatural amino acid thatcomprises a reactive functional group (de Graaf, A. J., et al. (2009)Bioconjug Chem 20: 1281-1295) that is used for covalent linkage with afunctionalized surfactant. By way of example, in some embodiments, alinker amino acid is Lys, or Orn having a reactive functional group—NH₂; or Cys, having a reactive functional group —SH; or Asp or Glu,having a reactive functional group —C(═O)—OH. By way of example, in someother embodiments, a linker amino acid is any amino acid having areactive functional group such as —OH, —N₃, haloacetyl or an acetylenicgroup that is used for formation of a covalent linkage with a suitablyfunctionalized surfactant.

As used herein, a “functionalized surfactant” is a surfactant comprisinga reactive group suitable for covalent linkage with a linker amino acid.By way of example, in some embodiments, a functionalized surfactantcomprises a carboxylic acid group (e.g., at the 6-position of amonosaccharide) as the reactive group suitable for covalent linkage witha linker amino acid. By way of example, in some embodiments, afunctionalized surfactant comprises a —NH₂ group, a —N₃ group, anacetylenic group, a haloacetyl group, a —O—NH₂ group, or a—(CH₂-)m-maleimide group, e.g., at the 6-position of a monosaccharide(as shown in Scheme 6), that allows for covalent linkage with a suitablelinker amino acid. In some embodiments, a functionalized surfactant is acompound of Formula II as described herein. Optionally, in some specificembodiments, a functionalized surfactant comprises a covalently attachedlinker amino acid; the surfactant-modified peptide is then formed bysequential addition of one or more amino acids to the linker amino acid.

As used herein, the term “peptide” is any peptide comprising two or moreamino acids. The term peptide includes polypeptides, short peptides(e.g., peptides comprising between 2-14 amino acids), medium lengthpeptides (15-50) or long chain peptides (e.g., proteins). The termspeptide, polypeptide, medium length peptide and protein may be usedinterchangeably herein. As used herein, the term “peptide” isinterpreted to mean a polymer composed of amino acid residues, relatednaturally occurring structural variants, and synthetic non-naturallyoccurring analogs thereof linked via peptide bonds, related naturallyoccurring structural variants, and synthetic non-naturally occurringanalogs thereof. Synthetic peptides can be synthesized, for example,using an automated peptide synthesizer.

Peptides may contain amino acids other than the 20 gene encoded aminoacids. “Peptide(s)” include those modified either by natural processes,such as processing and other post-translational modifications, but alsoby chemical modification techniques. Such modifications are welldescribed in basic texts and in more detailed monographs, and arewell-known to those of skill in the art. It will be appreciated that insome embodiments, the same type of modification is present in the sameor varying degree at several sites in a given peptide. Also, a givenpeptide, in some embodiments, contains more than one type ofmodifications. Modifications occur anywhere in a peptide, including thepeptide backbone, the amino acid side-chains, and the amino or carboxyltermini.

The term peptide includes peptides or proteins that comprise natural andunnatural amino acids or analogs of natural amino acids. As used herein,peptide and/or protein “analogs” comprise non-natural amino acids basedon natural amino acids, such as tyrosine analogs, which includespare-substituted tyrosines, ortho-substituted tyrosines, and metasubstituted tyrosines, wherein the substituent on the tyrosine comprisesan acetyl group, a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, a methyl group, anisopropyl group, a C₂-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a halogen, a nitro group, or the like. Examples of Tyr analogsinclude 2,4-dimethyl-tyrosine (Dmt), 2,4-diethyl-tyrosine,O-4-allyl-tyrosine, 4-propyl-tyrosine, Ca-methyl-tyrosine and the like.Examples of lysine analogs include ornithine (Orn), homo-lysine,Ca-methyl-lysine (CMeLys), and the like. Examples of phenylalanineanalogs include, but are not limited to, meta-substitutedphenylalanines, wherein the substituent comprises a methoxy group, aC₁-C₂₀ alkyl group, for example a methyl group, an allyl group, anacetyl group, or the like. Specific examples include, but are notlimited to, 2,4,6-trimethyl-L-phenylalanine (Tmt), O-methyl-tyrosine,3-(2-naphthyl)alanine (Nal(2)), 3-(1-naphthyl)alanine (Nal(1)),3-methyl-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic), fluorinated phenylalanines, isopropyl-phenylalanine,p-azido-phenylalanine, p-acyl-phenylalanine, p-benzoyl-phenylalanine,p-iodo-phenylalanine, p-bromophenylalanine, p-amino-phenylalanine, andisopropyl-phenylalanine, and the like. Other nonstandard or unnaturalamino acids used in peptide analog design include and are not limited toC-alpha-disubstituted amino acids such as Aib, Cα-diethylglycine (Deg),aminocyclopentane-1-carboxylic acid (Ac5c), and the like. Such aminoacids frequently lead to a restrained structure, often biased toward analpha helical structure (Kaul, R. and Balaram, P. (1999) Bioorg Med Chem7: 105-117). Additional examples of such unnatural amino acids useful inanalog design are homo-arginine (Har), and the like. Substitution ofreduced amide bonds in certain instances leads to improved protectionfrom enzymatic destruction or alters receptor binding. By way ofexample, incorporation of a Tic-Phe dipeptide unit with a reduced amidebond between the residues (designated as Tic-Ψ[CH2-NH]-Ψ-Phe) reducesenzymatic degradation. Accordingly, also contemplated within the scopeof embodiments described herein are surfactants covalently attached topeptides that comprise modified amino acids and/or peptide analogsdescribed above. Certain non-natural amino acids are shown below:

As used herein, the term “variant” is interpreted to mean a peptide thatdiffers from a reference peptide, but retains essential properties. Atypical variant of a peptide differs in amino acid sequence fromanother, reference peptide. Generally, differences are limited so thatthe sequences of the reference peptide and the variant are closelysimilar overall and, in many regions, identical. A variant and referencepeptide may differ in amino acid sequence by one or more substitutions,additions, deletions in any combination. A substituted or inserted aminoacid residue may or may not be one encoded by the genetic code.Non-naturally occurring variants of peptides may be made by mutagenesistechniques, by direct synthesis, and by other suitable recombinantmethods.

Methods

Provided herein, in some embodiments are methods for prevention and/ortreatment of conditions associated with decreases in insulin sensitivitycomprising administration of a therapeutically effective amount of asurfactant-modified peptide and/or protein product described herein(e.g., a peptide product of Formula I-A, III-A, III-B or Formula V) toindividuals in need thereof. In some embodiments, the conditionscharacterized by decreases in insulin sensitivity include, and are notlimited to, the metabolic syndrome, obesity-related insulin resistance,hypertension, systemic inflammation associated with high C reactiveprotein, diabetes, or the like.

Also provided herein are methods for treatment of insulin resistancecomprising administration of a therapeutically effective amount of asurfactant-modified peptide and/or protein product described herein(e.g., a peptide product of Formula I-A, III-A, III-B or Formula V) toindividuals in need thereof. In some embodiments, the insulin resistanceis associated with the metabolic syndrome (Syndrome X) and/or diabetes.

Further provided herein are methods for stimulating resensitization ofthe body to insulin comprising administration of a therapeuticallyeffective amount of a surfactant-modified peptide and/or protein productdescribed herein (e.g. a peptide product of Formula I-A, III-A, III-B orFormula V) to individuals in need thereof.

In yet further embodiments, provided herein are methods for increasinginsulin sensitivity through weight loss, comprising administration of atherapeutically effective amount of a surfactant-modified peptide and/orprotein product described herein (e.g. a peptide product of Formula I-A,III-A, III-B or Formula V and in Table 1 of FIG. 1 and Table 2 of FIG.2) to individuals in need thereof.

Also provided herein are methods of treating diabetes or prediabetescomprising administering to a subject in need thereof a therapeuticallyeffective amount of a peptide product described above and herein and inTable 1 of FIG. 1 and Table 2 of FIG. 2 to an individual in needthereof.

Provided herein are methods for treating or delaying the progression oronset of conditions selected from diabetes, diabetic retinopathy,diabetic neuropathy, diabetic nephropathy, insulin resistance,hyperglycemia, hyperinsulinemia, metabolic syndrome, diabeticcomplications, elevated blood levels of free fatty acids or glycerol,hyperlipidemia, obesity, hypertriglyceridemia, atherosclerosis, acutecardiovascular syndrome, infarction, ischemic reperfusion ahypertension, comprising administering a therapeutically effectiveamount of a peptide product described herein and in Table 1 of FIG. 1and Table 2 of FIG. 2 to an individual in need thereof. In an additionalembodiment, provided herein are methods for treating delays in woundhealing comprising administering a therapeutically effective amount of apeptide product described herein and in Table 1 of FIG. 1 and Table 2 ofFIG. 2 to an individual in need thereof.

In one embodiment said condition to be treated is diabetes. In oneembodiment said condition to be treated is insulin resistance. In oneembodiment said condition to be treated is the metabolic syndrome. Inone embodiment said effective amount of said peptide is from about 0.1μg/kg/day to about 100.0 μg/kg/day.

In one embodiment the method of administration is parenteral. In oneembodiment the method of administration is per oral. In one embodimentthe method of administration is subcutaneous. In one embodiment themethod of administration is nasal insufflation.

Further provided herein is a method of reducing weight gain or inducingweight loss comprising administering a therapeutically effective amountof a peptide product described herein and in Table 1 of FIG. 1 and Table2 of FIG. 2 to an individual in need thereof. In some embodiments, theweight gain is associated with metabolic syndrome.

Provided herein is a method of treating hypoglycemia comprisingadministering a therapeutically effective amount of a peptide productdescribed herein and in Table 1 of FIG. 1 and Table 2 of FIG. 2 to anindividual in need thereof.

Also provided herein are methods for treatment of diabetes comprisingadministering a therapeutically effective amount of a peptide productdescribed herein and in Table 1 of FIG. 1 and Table 2 of FIG. 2 to anindividual in need thereof and at least one additional therapeuticagent; wherein said therapeutic agent is selected from an antidiabeticagent, an anti-obesity agent, a satiety agent, an anti-inflammatoryagent, an anti-hypertensive agent, an anti-atherosclerotic agent and alipid-lowering agent.

In some embodiments of the methods described above, the peptide and/orprotein that is covalently attached to a surfactant is a glucagon orGLP-1 peptide, or an analog thereof. In some embodiments, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula I-A, III-A, III-B or Formula V) is administered prophylacticallyand delays occurrence of any condition associated with insulinresistance, including and not limited to the metabolic syndrome,hypertension, diabetes, type 2 diabetes, gestational diabetes,hyperlipidemia, atherosclerosis, systemic inflammation or the like. Insome embodiments, the surfactant-modified peptide and/or protein (e.g.,a peptide product of Formula I-A, III-A, III-B or Formula V) isadministered therapeutically and delays progression of any conditionassociated with the metabolic syndrome, hypertension, diabetes, type 2diabetes, gestational diabetes, hyperlipidemia, atherosclerosis,systemic inflammation or the like. In some embodiments, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula I-A, III-A, III-B or Formula V) is administered prophylacticallyand/or therapeutically and delays progression of insulin resistance todiabetes. In some embodiments, the surfactant-modified peptide and/orprotein (e.g., a peptide product of Formula I-A, III-A, III-B or FormulaV) is administered prophylactically and/or therapeutically and reducesor halts further loss of insulin resistance, thereby stabilizingdisease.

In some embodiments, the surfactant-modified peptide and/or protein(e.g., a peptide product of Formula I-A, III-A, III-B or Formula V) isadministered parenterally. In some embodiments, the surfactant-modifiedpeptide and/or protein (e.g., a peptide product of Formula I-A, III-A,III-B or Formula V) is administered subcutaneously. In some embodiments,the surfactant-modified peptide and/or protein (e.g., a peptide productof Formula I-A, III-A, III-B or Formula V) is administered by nasalinsufflation.

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula I-A, III-A, III-B or Formula V) has a longer duration of actioncompared to a pharmaceutical comprising currently known therapeutics(e.g., exenatide, metformin or the like).

Combination Therapy

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula I-A, III-A, III-B or Formula V) is administered in combinationwith other methods of treatment of the metabolic syndrome selected fromthe group comprising an antidiabetic agent, an anti-obesity agent, ananti-hypertensive agent, an anti-atherosclerotic agent and alipid-lowering agent. By way of example, efficacious antidiabetic agentssuitable for administration in combination with a surfactant-modifiedpeptide and/or protein product described herein include a biguanide, asulfonylurea, a glucosidase inhibitor a PPAR γ agonist, a PPAR α/γ dualagonist, an aP2 inhibitor, a DPP4 inhibitor, an insulin sensitizer, aGLP-1 analog, insulin and a meglitinide. Additional examples includemetformin, glyburide, glimepiride, glipyride, glipizide, chlorpropamide,gliclazide, acarbose, miglitol, pioglitazone, troglitazone,rosiglitazone, muraglitazar, insulin, GI-262570, isaglitazone, JTT-501,NN-2344, L895 645, YM-440, R-119702, A19677, repaglinide, nateglinide,KAD 1129, AR-HO 39242. GW-40 I 5 44, KRP2 I 7, AC2993, LY3 I 5902,NVP-DPP-728A and saxagliptin.

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula I-A, III-A, III-B or Formula V) is administered in combinationwith other methods of treatment of the metabolic syndrome selected fromthe group of efficacious anti-obesity agents. By way of example,efficacious anti-obesity agents suitable for administration with thepeptide products described herein include beta 3 adrenergic agonist, alipase inhibitor, a serotonin (and dopamine) reuptake inhibitor, athyroid receptor beta compound, a CB-1 antagonist, a NPY-Y2 and a NPY-Y4receptor agonist and an anorectic agent. Specific members of theseclasses comprise orlistat, AfL-962, A19671, L750355, CP331648,sibutramine, topiramate, axokine, dexamphetamine, phentermine,phenylpropanolamine, rimonabant (SR1 4I7164), and mazindol.

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula I-A, III-A, III-B or Formula V) is administered in combinationwith other methods of treatment of the metabolic syndrome selected fromthe group of efficacious lipid-lowering agents. By way of example,efficacious lipid-lowering agents suitable for administration with thepeptide products described herein include agents selected from the groupconsisting of an MTP inhibitor, cholesterol ester transfer protein, anHMG CoA reductase inhibitor, a squalene synthetase inhibitor, a fibricacid derivative, an upregulator of LDL receptor activity, a lipoxygenaseinhibitor, and an ACAT inhibitor. Specific examples from these classescomprise pravastatin, lovastatin, simvastatin, atorvastatin,cerivastatin, fluvastatin, nisvastatin, visastatin, fenofibrate,gemfibrozil, clofibrate, avasimibe, TS-962, MD-700, CP-52941 4, andLY295 427.

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula I-A, III-A, III-B or Formula V) is administered in combinationwith peptide hormones, and analogs thereof, that are known to exhibitpro-satiety effects in animal models and in man. Contemplated within thescope of embodiments presented herein is a combination of the peptideproducts described herein and long-acting satiety agents for treatmentof obesity. Examples of such peptide satiety agents include GLP-1,pancreatic polypeptide (PP), cholecystokinin (CCK), peptide YY (PYY),amylin, calcitonin, OXM, neuropeptide Y (NPY), and analogs thereof(Bloom, S. R., et al. (2008) Mol Interv 8: 82-98; Field, B. C., et al.(2009) Br J Clin Pharmacol 68: 830-843).

Also contemplated within the scope of embodiments presented herein aremethods for treatment of obesity comprising administration of peptideproducts described herein in combination with peptide hormones includingand not limited to leptin, ghrelin and CART (cocaine- andamphetamine-regulated transcript) analogs and antagonists.

Additional peptide products in the body are associated with fat cells orthe obese state (adipokines) and are known to have proinflammatoryeffects (Gonzalez-Periz, A. and Claria, J. (2010) ScientificWorldJournal10: 832-856). Such agents will have additional favorable actions whenused in combination with the peptide products described herein. Examplesof agents that offer a beneficial effect when used in combination withthe peptide products described herein include analogs and antagonists ofadiponectin, chemerin, visfatin, nesfatin, omentin, resistin, TNFalpha,IL-6 and obestatin.

Intermediates

In one embodiment the provided herein are intermediates and/or reagentscomprising a surfactant moiety and a reactive functional group capableof forming a bond with a reactive functional group on a natural orunnatural amino acid. These intermediates and/or reagents allow forimprovement in the bioavailability and pharmaceutical, pharmacokineticand/or pharmacodynamic behavior of peptides and/or proteins of use inhuman and animal disease. Covalent attachment of such intermediatesand/or reagents via functional group on a side chain of an amino acid,for example on an epsilon-amino function of Lys, the sulfhydryl of Cys,or at the amino or carboxy terminus of the peptide and/or protein targetallows for synthesis of the peptide products described herein. Inspecific embodiments, non-ionic surfactant moieties are mono ordisaccharides with an O-alkyl glycosidic substitution, said glycosidiclinkage being of the alpha or beta configuration. In specificembodiments, O-alkyl chains are from C₁-C₂₀ or from C₆-C₁₆ alkyl chains.

In another embodiment provided herein are intermediates and/or reagentscomprising a non-ionic surfactant moiety with certain alkyl glycosidiclinkage that mimic O-alkyl glycosidic linkages and a reactive functionalgroup capable of forming a bond with a reactive functional group on anatural or unnatural amino acid. Such intermediates and/or reagentscontain S-linked alkyl chains or N-linked alkyl chains and have alteredchemical and/or enzymatic stability compared to the O-linked alkylglycoside-linked products.

In some embodiments, an intermediate and/or reagent provided herein is acompound wherein the hydrophilic group is a modified glucose, galactose,maltose, glucuronic acid, diglucuronic acid or the like. In someembodiments, the hydrophilic group is glucose, maltose, glucuronic acid,or diglucuronic acid and the hydrophobic group is a C₁-C₂₀ alkyl chainor an aralkyl chain. In some embodiments the glycosidic linkage to thehydrophobic group is of an alpha configuration and in some the linkageis beta at the anomeric center on the saccharide.

In some embodiments, the hydrophilic group is glucose, maltose,glucuronic acid, or diglucuronic acid and the hydrophobic group is aC₁-C₂₀ alkyl or aralkyl chain.

In some embodiments, an intermediate and/or reagent provided hereincomprises a surfactant containing a reactive functional group that is acarboxylic acid group, an amino group, an azide, an aldehyde, amaleimide, a sulfhydryl, a hydroxylamino group, an alkyne or the like.

In some embodiments, the intermediate and/or reagent is an O-linkedalkyl glycoside with one of the hydroxyl functions modified to be acarboxylic acid or amino functional group.

In some embodiments, the reagent is a 1-O-alkyl glucuronic acid of alphaor beta configuration and the alkyl chain is from C₁ to C₂₀ in length.In some of such embodiments, the alkyl group is from C₆ to C₁₆ inlength.

In some embodiments, the reagent comprises a 1-O-alkyl diglucuronic acidof alpha or beta configuration and the alkyl chain is from C₁ to C₂₀ inlength. In some of such embodiments, the alkyl group is from C₆ to C₁₆in length.

In some embodiments, the reagent is an S-linked alkyl glycoside of alphaor beta configuration with one of the hydroxyl functions modified to bea carboxylic acid or amino functional group.

In some embodiments, the reagent is an N-linked alkyl glycoside of alphaor beta configuration with one of the hydroxyl functions modified to bea carboxylic acid or amino functional group.

In yet another embodiment the provided herein are peptide and/or proteinproducts containing a covalently linked alkyl glycoside with propertiesacceptable for use in human and animal disease. Scheme 1 lists exemplarynon-ionic surfactants that can be modified to yield the reagents and/orintermediates that are useful for synthesis of surfactant-modifiedpeptide products described herein.

In some embodiments, the covalently modified peptides and/or proteinsdescribed herein incorporate a surfactant moiety into the peptidestructure. In specific embodiments, the covalently modified peptidesand/or proteins described herein incorporate a non-ionic surfactant ofthe alkyl, alkoxyaryl, or aralkyl glycoside class. Alkyl glycosides areimportant commodities and are widely used in the food, service andcleaning industries. Thus their production on commercially significantscale has been the subject of extensive study. Both enzymatic andchemical processes are available for their production at very low cost(Park, D. W., et al. (2000) Biotechnology Letters 22: 951-956). Thesealkyl glycosides can be modified further to generate the intermediatesfor the synthesis of the covalently modified peptides and/or proteinsdescribed herein. Thus it is known that 1-dodecyl beta-D-glucoside ispreferentially oxidized on the 6-position to yield the correspondingglucuronic acid analog in high yield when using the unprotected materialand platinum black catalyst in the presence of oxygen (van Bekkum, H.(1990) Carbohydrates as Organic Raw Materials 289-310). Additionalchemoselective methods for oxidation of the primary alcohol at the 6position of alkyl glucosides are available. For example, use ofcatalytic amounts of 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) withstoichiometric amounts of the organic oxidant [bis(acetoxy)iodo]benzene(BAIB) (De Mico, A., et al. (1997) J Org Chem 1997: 6974-6977) gaveoutstanding yields of nucleoside-5′-carboxylic acids (Epp, J. B. andWidlanski, T. S. (1999) J Org Chem 64: 293-295) by oxidation of theprimary hydroxyl. This oxidation is chemoselective for the primaryhydroxyl even when the other, secondary hydroxyls are unprotected(Codee, J. D., et al. (2005) J Am Chem Soc 127: 3767-3773). In a similarmanner, 1-dodecyl 3-D-glucopyranoside, 1-tetradecyl β-D-glucopyranoside,1-hexadecyl β-D-glucopyranoside, 1-octadecyl-D-glucopyranoside and1-eicosyl β-D-glucopyranoside were oxidized to the corresponding uronicacids (1-dodecyl β-D-glucuronic acid, 1-tetradecyl β-D-glucuronic acid,1-hexadecyl β-D-glucuronic acid, 1-octadecyl β-D-glucuronic acid,1-eicosyl β-D-glucuronic acid) by oxidation with TEMPO using KBr andsodium hypochlorite as stoichiometric oxidant (Milkereit, G., et al.(2004) Chem Phys Lipids 127: 47-63) in water. A mild oxidation procedureusing (diacetoxyiodo)benzene (DAIB aka BAIB) is given in the Examples.Certain of these glucuronic acid intermediates are commerciallyavailable (for example octyl b-D-glucuronic acid; Carbosynth, Mo. 07928)and, as indicated, a broad range are subject to preparation by routinemethods (Schamann, M. and Schafer, H. J. (2003) Eur J Org Chem 351-358;Van den Bos, L. J., et al. (2007) Eur J Org Chem 3963-3976) or, uponrequest, from commercial sources. Scheme 2 illustrates, as examples,certain functionalized surfactant intermediates comprising a —COOH groupas a reactive functional group that are used to prepare theintermediates and/or reagents described herein.

Similarly, aralkyl glycosides (including alkoxyaryl) can form the basisfor closely related nonionic surfactant reagents. For example,4-alkoxyphenyl β-D-glucopyranosides are readily synthesized by thereaction of 4-alkyloxyphenols with penta-O-acetyl β-D-glucose in thepresence of boron trifluoride etherate. Subsequent deacetylation usingtrimethylamine in methanol/water and selective oxidation as describedabove and in the examples, yields the alkoxyaryl glucuronic acidreagents suitable for forming the reagents and peptides described herein((Smits, E., et al. (1996) J Chem Soc, Perkin Trans I 2873-2877; Smits,E., et al. (1997) Liquid Crystals 23: 481-488).

The glucuronic acid class of intermediate is readily activated bystandard coupling agents for linkage to an amino acid side chain, e.g.that of Lys. Thus Fmoc-Lys-O-TMS (trimethylsilyl=TMS) can be reactedwith octyl beta-D-glucuronic acid in the presence of a coupling agentand the O-TMS protecting group can then be hydrolyzed on aqueous workupto yield Fmoc-Lys(1-octyl bcta-D-glucuronamide) as shown in Scheme 4.This reagent can be used for incorporation into the solid phasesynthesis of peptides, using standard coupling protocols, when it isdesired to incorporate the surfactant moiety near the N-terminal regionof the molecule. The secondary hydroxyl groups can be left unprotected,due to the very much higher reactivity of the Lys amino functional groupor they can be protected by peracetylation. If an acetyl protected formis used, the acetyl protecting groups can be removed in high yield bytreatment with either MeOH/NaOMe or by MeOH/Et₃N. Scheme 4 illustratespreparation of the reagents described herein.

In some embodiments, reagents and/or intermediates for the preparationof the biologically active peptide products described herein comprise afamily of surfactant-modified linker amino acids for incorporation intosynthetic peptide products. Thus in one embodiment, peptide productsdescribed herein are synthesized in a linear fashion wherein afunctionalized surfactant is attached to a reversibly-protected linkeramino acid via functional group on a side chain of a linker amino acid(e.g., an amino group of a lysine residue) to yield a proprietaryreagent (as shown in Scheme 4.) which can be incorporated into thegrowing peptide chain and then the remaining peptide is synthesized byattachment of further amino acids to the cysteine residue. Protectinggroup suitable for synthesis of modified peptides and/or proteindescribed herein are described in, for example, T. W. Green, P. G. M.Wuts, Protective Groups in Organic Synthesis, Wiley-Interscience, NewYork, 1999, 503-507, 736-739, which disclosure is incorporated herein byreference.

In another embodiment, peptide products described herein are synthesizedby covalent attachment of a functionalized surfactant to a full-lengthpeptide via suitable functional group on a linker amino acid that is inthe peptide chain.

Alternatively a functionalized surfactant can be added to a linker aminoacid side chain which has been deprotected during the course of thesolid phase synthesis of the peptide. As an example, an alkyl glucuronylgroup can be added directly to a linker amino acid side chain (e.g., adeprotected Lys side chain) during the solid phase synthesis of thepeptide. For example, use of Fmoc-Lys(Alloc)-OH as a subunit providesorthogonal protection that can be removed while the peptide is still onthe resin. Thus deprotection of the Lys side chain using Pd/thiobarbitalor other Alloc deprotection recipe allows exposure of the amino groupfor coupling with the acyl protected or unprotected 1-octylbeta-D-glucuronic acid unit. Final deprotection with a low % CF₃CO₂H(TFA) cleavage cocktail will then deliver the desired product. Althoughthe glycosidic linkage is labile to strong acid, the experience here andby others is that it is relatively stable to low % TFA cleavageconditions. Alternatively, acyl protection (e.g. acetyl, Ac; benzoyl,Bz) or trialkylsilyl protection on the saccharide OH functional groupsmay be used to provide increased protection to the glycosidic linkage.Subsequent deprotection by base (NH₂NH₂/MeOH; NH₃/MeOH, NaOMe/MeOH)yields the desired deprotected product. Scheme 4 illustrates reagentsdescribed herein. Scheme 5 illustrates a non-limiting example of apeptide intermediate described herein. Although this example illustratesa peptide with the surfactant linkage at the N-terminus of the peptide,the methods described herein are suitable for synthesis of peptideintermediates having the linkage to a surfactant in the middle region,the C-terminal region or any position within the peptide.

Additional reagents are generated by modification of the 6-positionfunctional group to give varied means of linkage to amino acid sidechain functional groups, as shown below in Scheme 6. Thus aminosubstitution can be used for linkage to Asp or Glu side chains. Azido oralkyne substitution can be used for linkage to unnatural amino acidscontaining the complementary acceptor for Huisgen 3+2 cycloaddition(Gauthier, M. A. and Klok, H. A. (2008) Chem Commun (Camb) 2591-2611).Aminoxy or aldehyde functional groups can be used to link to aldehyde(i.e. oxime linkage) or to amino functions (i.e. reductive alkylation),respectively. The maleimide or —NH—(C═O)—CH₂—Br functional group canbind chemoselectively with a Cys or other SH functional group. Thesetypes of linkage strategies are advantageous when used in conjunctionwith the reagents described herein. Interconversion of functional groupsis widely practiced in organic synthesis and comprehensive lists ofmultiple routes to each of the functional group modifications listedherein are available (Larock, R. C. (1999)) “Comprehensive OrganicTransformations”, VCH Publishers, New York.

Thus, for example, the primary hydroxyl on position 6 of octyl1-β-D-glucoside is converted to the azide by activation and displacementwith an azide anion, reactions such as reactions used in carbohydratechemistry (e.g. by tosylation followed by NaN₃). The corresponding azideis reduced to the amino function by reduction with thiolacetic acid inpyridine (Elofsson, M., et al. (1997) Tetrahedron 53: 369-390) or bysimilar methods of amino group generation (Stangier, P., et al. (1994)Liquid Crystals 17: 589-595). Approaches to the acetylene, aminoxy, andaldehyde moieties are best carried out on the triacetoxy form, availablefrom the commercially available glucoside by treatment with Ac₂O,followed by mild hydrolysis of the primary amine. This 6-hydroxy formcan be selectively oxidized to the aldehyde, or activated as a tosylateor triflate and displaced by NH₂OH or by sodium acetylide. The maleimidelinkage can be through a carbon linkage as shown or, preferably thoughan O or amide linkage, again by displacement of the activated hydroxylor coupling of the glucuronic acid derivative to an amino linkedmaleimide reagent, well known in the art. Additional functional groupinterconversions are well known to those of average skill in the art ofmedicinal chemistry and are within the scope of the embodimentsdescribed herein.

Also contemplated within the scope of synthetic methods described hereinare surfactants wherein the saccharide and hydrophobic chain arecovalently attached via an alpha glycosidic linkage. Synthetic routes topredominantly α-linked glycosides are well known in the art andtypically originate with the peracetyl sugar and use acidic catalysis(e.g. SnCl₄, BF₃ or HCl) to effect the α-glycosylation (Cudic, M. andBurstein, G. D. (2008) Methods Mol Biol 494: 187-208; Vill, V., et al.(2000) Chem Phys Lipids 104: 75-91, incorporated herein by reference forsuch disclosure). Similar synthetic routes exist for disaccharideglycosides (von Minden, H. M., et al. (2000) Chem Phys Lipids 106:157-179, incorporated herein by reference for such disclosure).Functional group interconversions then proceed as above to lead to the6-carboxylic acid, et al. for generation of the corresponding α-linkedreagents.

Scheme 6 lists certain compounds and reagents useful in the synthesis ofthe covalently modified peptides and/or proteins described herein.Standard nomenclature using single letter abbreviations for amino acidsare used.

Many alkyl glycosides can be synthesized by known procedures, asdescribed, e.g., in (Rosevear, P., et al. (1980) Biochemistry 19:4108-4115, Li, Y. T., et al. (1991) J Biol Chem 266: 10723-10726) orKoeltzow and Urfer, J. Am. Oil Chem. Soc., 61:1651-1655 (1984), U.S.Pat. Nos. 3,219,656 and 3,839,318 or enzymatically, as described, e.g.,in (Li, Y. T., et al. (1991) J Biol Chem 266: 10723-10726, Gopalan, V.,et al. (1992) J Biol Chem 267: 9629-9638). O-alkyl linkages to naturalamino acids such as Ser can be carried out on the Fmoc-Ser-OH usingperacetylglucose to yieldNu-Fmoc-4-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-L-serine. Thismaterial is selectively deprotected at the primary carbon atom (position6) and selectively oxidized using TEMPO/BAIB as described above to yieldthe corresponding 6-carboxyl function which may be coupled to lipophilicamines to generate a new class of nonionic surfactant and reagents(Scheme 7).

The linkage between the hydrophobic alkyl and the hydrophilic saccharidecan include, among other possibilities, a glycosidic, thioglycosidic,amide (Carbohydrates as Organic Raw Materials, F. W. Lichtenthaler ed.,VCH Publishers, New York, 1991), ureido (Austrian Pat. 386,414 (1988);Chem. Abstr. 110:137536p (1989); see Gruber, H. and Greber, G.,“Reactive Sucrose Derivatives” in Carbohydrates as Organic RawMaterials, pp. 95-116) or ester linkage (Sugar Esters: Preparation andApplication, J. C. Colbert ed., (Noyes Data Corp., New Jersey), (1974)).

Examples from which useful alkyl glycosides can be chosen formodification to the reagents or for the formulation of the productsdescribed herein, include: alkyl glycosides, such as octyl-, nonyl-,decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-,hexadecyl-, heptadecyl-, and octadecyl-D-maltoside, -glucoside or-sucroside (i.e., sucrose ester) (synthesized according to Koeltzow andUrfer; Anatrace Inc., Maumee, Ohio; Calbiochem, San Diego, Calif.; FlukaChemie, Switzerland); alkyl thiomaltosides, such as heptyl, octyl,dodecyl-, tridecyl-, and tetradecyl-(β-D-thiomaltoside (synthesizedaccording to Defaye, J. and Pederson, C., “Hydrogen Fluoride, Solventand Reagent for Carbohydrate Conversion Technology” in Carbohydrates asOrganic Raw Materials, 247-265 (F. W. Lichtenthaler, ed.) VCHPublishers, New York (1991); Ferenci, T., J. Bacteriol, 144:7-11(1980)); alkyl thioglucosides, such as 1-dodecyl- or 1-octyl-thio α-orβ-D-glucopyranoside (Anatrace, Inc., Maumee, Ohio; see Saito, S. andTsuchiya, T. Chem. Pharm. Bull. 33:503-508 (1985)); alkyl thiosucroses(synthesized according to, for example, Binder, T. P. and Robyt, J. F.,Carbohydr. Res. 140:9-20 (1985)); alkyl maltotriosides (synthesizedaccording to Koeltzow and Urfer); long chain aliphatic carbonic acidamides of sucrose amino-alkyl ethers; (synthesized according to AustrianPatent 382,381 (1987); Chem. Abstr., 108:114719 (1988) and Gruber andGreber pp. 95-116); derivatives of palatinose and isomaltamine linked byamide linkage to an alkyl chain (synthesized according to Kunz, M.,“Sucrose-based Hydrophilic Building Blocks as Intermediates for theSynthesis of Surfactants and Polymers” in Carbohydrates as Organic RawMaterials, 127-153); derivatives of isomaltamine linked by urea to analkyl chain (synthesized according to Kunz); long chain aliphaticcarbonic acid ureides of sucrose amino-alkyl ethers (synthesizedaccording to Gruber and Greber, pp. 95-116); and long chain aliphaticcarbonic acid amides of sucrose amino-alkyl ethers (synthesizedaccording to Austrian Patent 382,381 (1987), Chem. Abstr., 108:114719(1988) and Gruber and Greber, pp. 95-116).

Some preferred glycosides which can be further modified to incorporatereactive functionality for linkage to the peptide include thesaccharides maltose, sucrose, glucose and galactose linked by glycosidicor ester linkage to an alkyl chain of 6, 8, 10, 12, 14, or 16 carbonatoms, e.g., hexyl-, octyl-, decyl-, dodecyl-, tetradecyl-, andhexadecyl-maltoside, sucroside, glucoside and galactoside. In the bodythese glycosides are degraded to non-toxic alcohol or fatty acid and anoligosaccharide or saccharide. The above examples are illustrative ofthe types of alkyl glycosides to be used in the methods claimed herein,however the list is not intended to be exhaustive.

Generally, these surfactants (e.g., alkyl glycosides) are optionallydesigned or selected to modify the biological properties of the peptide,such as to modulate bioavailability, half-life, receptor selectivity,toxicity, biodistribution, solubility, stability, e.g. thermal,hydrolytic, oxidative, resistance to enzymatic degradation, and thelike, facility for purification and processing, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic activity, redox potential, ability to react with othermolecules, e.g., covalently or noncovalently, and the like.

Surfactants

The term “surfactant” comes from shortening the phrase “surface activeagent”. In pharmaceutical applications, surfactants are useful in liquidpharmaceutical formulations in which they serve a number of purposes,acting as emulsifiers, solubilizers, and wetting agents. Emulsifiersstabilize the aqueous solutions of lipophilic or partially lipophilicsubstances. Solubilizers increase the solubility of components ofpharmaceutical compositions increasing the concentration which can beachieved. A wetting agent is a chemical additive which reduces thesurface tension of a fluid, inducing it to spread readily on a surfaceto which it is applied, thus causing even “wetting” of the surface withthe fluids. Wetting agents provide a means for the liquid formulation toachieve intimate contact with the mucous membrane or other surface areaswith which the pharmaceutical formulation comes in contact. Thussurfactants may be useful additives for stabilization of the formulationof the peptide products described herein as well as for the modificationof the properties of the peptide itself.

In specific embodiments, alkyl glycosides which are syntheticallyaccessible, e.g., the alkyl glycosides dodecyl, tridecyl and tetradecylmaltoside or glucoside as well as sucrose dodecanoate, tridecanoate, andtetradecanoate are suitable for covalent attachment to peptides asdescribed herein. Similarly, the corresponding alkylthioglucosides arestable, synthetically accessible surfactants which are acceptable forformulation development.

A wide range of physical and surfactant properties can be achieved byappropriate modification of the hydrophobic or hydrophilic regions ofthe surfactant (e.g., the alkyl glycoside). For example, a studycomparing the bilayer activity of dodecyl maltoside (DM) with that ofdodecyl glucoside (DG) found that of DM to be more than three timeshigher than that of DG, despite having the same length of hydrophobictail (Lopez, O., et al. (2002) Colloid Polym Sci 280: 352-357). In thisparticular instance the identity of the polar region (disaccharide vs.monosaccharide) influences surfactant behavior. In the case of asurfactant linked to a peptide, e.g. the peptide products describedherein, the peptide region also may contribute hydrophobic orhydrophilic character to the overall molecule. Thus tuning of thephysical and surfactant properties may be used to achieve the particularphysical and pharmaceutical properties suitable for the individualpeptide targets.

PEG Modification

In some embodiments, surfactant-modified peptide products describedherein are further modified to incorporate one or more PEG moieties(Veronese, F. M. and Mero, A. (2008) BioDrugs 22: 315-329). In someinstances, incorporation of large PEG chains prevents filtration of thepeptide in the glomeruli in the kidney into the dilute urine formingthere (Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:4399-4418, Caliceti, P. and Veronese, F. M. (2003) Adv Drug Deliv Rev55: 1261-1277). In some embodiments, an optional PEG hydrophilic chainallows for balancing the solubility and physical properties of thepeptides or proteins that have been rendered hydrophobic by theincorporation of the longer chain alkyl glycoside moiety.

PEGylation of a protein can have potentially negative effects as well.Thus PEGylation can cause a substantial loss of biological activity forsome proteins and this may relate to ligands for specific classes ofreceptors. In such instances there may be a benefit to reversiblePEGylation (Peleg-Shulman, T., et al. (2004) J Med Chem 47: 4897-4904,Greenwald, R. B., et al. (2003) Adv Drug Deliv Rev 55: 217-250, Roberts,M. J. and Harris, J. M. (1998) J Pharm Sci 87: 1440-1445).

In addition, the increased molecular mass may prevent penetration ofphysiological barriers other than the glomerular membrane barrier. Forexample, it has been suggested that high molecular weight forms ofPEGylation may prevent penetration to some tissues and thereby reducetherapeutic efficacy. In addition, high molecular weight may preventuptake across mucosal membrane barriers (nasal, buccal, vaginal, oral,rectal, lung delivery). However delayed uptake may be highlyadvantageous for administration of stable molecules to the lung,substantially prolonging the duration of action. The peptide and/orprotein products described herein have increased transmucosalbioavailability and this will allow longer chain PEG modifications to beused in conjunction with the surfactant modification with theachievement of commercially significant bioavailability followingintranasal or other transmucosal route.

In some embodiments, long chain PEG polymers, and short chain PEGpolymers are suitable for modification of the proteins and peptidesdescribed herein. Administration of treatments for diabetes byinhalation is a new approach for drug delivery and the lung has a highlypermeable barrier (e.g. Exubera). For this application, delayedpenetration of the lung barrier, preferred forms of PEGylation are inthe lower molecular weight range of C₁₀ to C₄₀₀ (roughly 250 to 10,000Da). Thus while a primary route to prolongation by PEG is theachievement of an “effective molecular weight” above the glomerularfiltration cut-off (greater than 68 kDa), use of shorter chains may be aroute for prolongation of residence in the lung for treatment of lungdiseases and other respiratory conditions. Thus PEG chains of about 500to 3000 Da are of sufficient size to slow the entry into the peripheralcirculation, but insufficient to cause them to have a very prolongedcirculation time. In some embodiments, PEGylation is applied to giveincreased local efficacy to the lung tissue with reduced potential forsystemic side effects for the covalently modified peptides and/orproteins described herein. In some of such embodiments, PEG chains inthe range from about 750 to about 1500 Da are referred collectively as“PEG1K.”

In addition, other polymers may be used in conjunction with thecompounds of described herein in order to optimize their physicalproperties. For example poly(2-ethyl 2-oxazoline) conjugates havevariable hydrophobicity and sufficient size to enhance duration ofaction (Mero, A., et al. (2008) J Control Release 125: 87-95). Linkageof such a polymer to a saccharide yields a class of surfactant suitablefor use in modification of peptides and/or proteins described herein.

Polyethylene glycol chains are functionalized to allow their conjugationto reactive groups on the peptide and/or protein chain. Typicalfunctional groups allow reaction with amino, carboxyl or sulfhydrylgroups on the peptide through the corresponding carboxyl, amino ormaleimido groups (and the like) on the polyethylene glycol chain. In anembodiment, PEG comprises a C₁₀-C₃₀₀₀ chain. In another embodiment, PEGhas a molecular weight above 40,000 Daltons. In yet another embodiment,PEG has a molecular weight below 10,000 Daltons. PEG as a proteinmodification is well known in the art and its use is described, forexample, in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192; and 4,179,337.

A non-traditional type of PEG chain is modified to be amphiphilic innature. That is it has both the hydrophilic PEG structure but ismodified to contain hydrophobic regions such as fatty acid esters andother hydrophobic components. See for example (Miller, M. A., et al.(2006) Bioconjug Chem 17: 267-274); Ekwuribe, et al. U.S. Pat. No.6,309,633; Ekwuribe, et al. U.S. Pat. No. 6,815,530; Ekwuribe, et al.U.S. Pat. No. 6,835,802). Although these amphiphilic PEG conjugates toproteins were originally developed to increase oral bioavailability theywere relatively ineffective in this role. However the use of suchamphiphilic PEG conjugates with amphipathic peptides will givesignificantly prolonged residence in the lung to extend the usefulbiological activity of these pharmaceuticals. The preferred PEG chainsare in the molecular weight range of 500 to 3000 Da. Detaileddescriptions of the methods of synthesis of these conjugates is given inthe references above, the full content of which is incorporated herein.

A PEG entity itself does not have a functional group to be attached to atarget molecular, such as a peptide. Therefore, to create PEGattachment, a PEG entity must be functionalized first, then afunctionalized attachment is used to attach the PEG entity to a targetmolecule, such as a peptide (Greenwald, R. B., et al. (2003) Adv DrugDeliv Rev 55: 217-250, Veronese, F. M. and Pasut, G. (2005) Drug DiscovToday 10: 1451-1458, Roberts, M. J., et al. (2002) Adv Drug Deliv Rev54: 459-476). In one embodiment, site-specific PEGylation can beachieved through Cys substitution on a peptide molecule. The targetpeptide can be synthesized by solid phase synthesis, recombinant means,or other means, as described herein.

Thus in some embodiments, a peptide product described herein comprises aLys or other reactive residue modified with an alkyl glycoside andspecific PEGylation on at least one Cys residue, a Lys residue or otherreactive amino acid residue elsewhere in the molecule.

In another embodiment, a Lys or other residue with a nucleophilic sidechain may be used for incorporation of the PEG residue. This may beaccomplished through the use of an amide or carbamate linkage to aPEG-carboxyl or PEG-carbonate chain. See for example as described(Veronese, F. M. and Pasut, G. (2005) Drug Discov Today 10: 1451-1458).An alternative approach is to modify the Lys side chain amino functionthrough attachment of an SH containing residue, such as mercaptoacetyl,mercaptopropionyl (CO—CH2-CH2-CH2-SH), and the like. Alternatively thePEG chain may be incorporated at the C-Terminus as an amide during thecourse of the synthesis. Additional methods for attaching PEG chainsutilize reaction with the side chains of His and Trp. Other similarmethods of modifying the peptide chain to allow attachment of a PEGchain are known in the art and are incorporated herein by reference(Roberts, M. J., et al. (2002) Adv Drug Deliv Rev 54: 459-476).

Formulations

In one embodiment, the covalently modified peptides or proteins asdisclosed herein are provided in a formulation that further reduces,prevents, or lessens peptide and/or protein association or aggregationin the composition, for example, reduces peptide and/or proteinself-association or self-aggregation, or reduces association oraggregation with other peptides or proteins when administered to thesubject.

Self-Association at high protein concentration is problematic intherapeutic formulations. For example, self-association increases theviscosity of a concentrated monoclonal antibody in aqueous solution.Concentrated insulin preparations are inactivated by self aggregation.These self associating protein interactions, particularly at highprotein concentration, reduce, modulate or obliterate biologicalactivity of many therapeutics (Clodfelter, D. K., et al. (1998) PharmRes 15: 254-262). Therapeutic proteins formulated at high concentrationsfor delivery by injection or other means can be physically unstable orbecome insoluble as a result of these protein interactions.

A significant challenge in the preparation of peptide and proteinformulations is to develop manufacturable and stable dosage forms.Physical stability properties, critical for processing and handling, areoften poorly characterized and difficult to predict. A variety ofphysical instability phenomena are encountered such as association,aggregation, crystallization and precipitation, as determined by proteininteraction and solubility properties. This results in significantmanufacturing, stability, analytical, and delivery challenges.Development of formulations for peptide and protein drugs requiring highdosing (on the order of mg/kg) are required in many clinical situations.For example, using the SC route, approximately <1.5 mL is the allowableadministration volume. This may require >100 mg/mL proteinconcentrations to achieve adequate dosing. Similar considerations existin developing a high-concentration lyophilized formulation formonoclonal antibodies. In general, higher protein concentrations permitsmaller injection volume to be used which is very important for patientcomfort, convenience, and compliance. The surfactant-modified compoundsdescribed herein are designed to minimize such aggregation events andmay be further facilitated through the use of small amounts ofsurfactants as herein described.

Because injection is an uncomfortable mode of administration for manypeople, other means of administering peptide therapeutics have beensought. Certain peptide and protein therapeutics may be administered,for example, by intranasal, buccal, oral, vaginal, inhalation, or othertransmucosal administration. Examples are nafarelin (Synarel®) andcalcitonin which are administered as commercial nasal sprayformulations. The covalently modified peptides and/or proteins describedherein are designed to facilitate such transmucosal administration andsuch formulations may be further facilitated through the use of smallamounts of surfactants as described herein.

Typical formulation parameters include selection of optimum solution pH,buffer, and stabilizing excipients. Additionally, lyophilized cakereconstitution is important for lyophilized or powdered formulations. Afurther and significant problem comprises changes in viscosity of theprotein formulation upon self-association. Changes in viscosity cansignificantly alter delivery properties e.g., in spray (aerosol)delivery for intranasal, pulmonary, or oral cavity sprays. Furthermore,increased viscosity can make injection delivery by syringe or iv linemore difficult or impossible.

Many attempts to stabilize and maintain the integrity and physiologicalactivity of peptides have been reported. Some attempts have producedstabilization against thermal denaturation and aggregation, particularlyfor insulin pump systems. Polymeric surfactants are described (Thurow,H. and Geisen, K. (1984) Diabetologia 27: 212-218; Chawla, A. S., et al.(1985) Diabetes 34: 420-424). The stabilization of insulin by thesecompounds was believed to be of a steric nature. Among other systemsused are saccharides (Arakawa, T. and Timasheff, S. N. (1982)Biochemistry 21: 6536-6544), osmolytes, such as amino acids (Arakawa, T.and Timasheff, S. N. (1985) Biophys J 47: 411-414), and water structurebreakers, such as urea (Sato, S., et al. (1983) J Pharm Sci 72:228-232). These compounds exert their action by modulating theintramolecular hydrophobic interaction of the protein or peptide.

Various peptides, peptides, or proteins are described herein and may bemodified with any of the covalently bound surfactant reagents describedherein. Advantageously, the peptide modifications described hereincomprise covalent attachment of a surfactant that comprises bothhydrophilic (e.g. saccharide) and hydrophobic (e.g. alkyl chain) groups,thereby allowing for stabilization of the peptide in physiologicalconditions. In some embodiments, covalent linkage of a moiety comprisinga hydrophilic group and hydrophobic group (e.g. a glycoside surfactant)to a peptide, and/or protein described herein eliminates the need formodifying the amino acid sequence of the peptide, and/or protein toenhance stability (e.g., reduce aggregation).

In some embodiments, the formulations comprise at least one drugcomprising a peptide modified with a surfactant derived reagentdescribed herein and in formulation additionally may be associated witha surfactant, wherein the surfactant is further comprised of, forexample, a saccharide, an alkyl glycoside, or other excipient and can beadministered in a format selected from the group consisting of a drop, aspray, an aerosol, a lyophilizate, a spray dried product, an injectable,and a sustained release format. The spray and the aerosol can beachieved through use of the appropriate dispenser and may beadministered by intranasal, transbuccal, inhalation or othertransmucosal route. The lyophilizate may contain other compounds such asmannitol, saccharides, submicron anhydrous α-lactose, gelatin,biocompatible gels or polymers. The sustained release format can be anocular insert, erodible microparticulates, hydrolysable polymers,swelling mucoadhesive particulates, pH sensitive microparticulates,nanoparticles/latex systems, ion-exchange resins and other polymericgels and implants (Ocusert, Alza Corp., California; Joshi, A., S. Pingand K. J. Himmelstein, Patent Application WO 91/19481). Significant oralbioavailability is also achievable.

The peptide and protein modifications described herein mitigate and, insome cases, may eliminate the need for organic solvents. Trehalose,lactose, and mannitol and other saccharides have been used to preventaggregation. Aggregation of an anti-IgE humanized monoclonal antibodywas minimized by formulation with trehalose at or above a molar ratio inthe range of 300:1 to 500:1 (excipient:protein). However, the powderswere excessively cohesive and unsuitable for aerosol administration orexhibited unwanted protein glycation during storage (Andya, J. D., etal. (1999) Pharm Res 16: 350-358). Each of the additives discovered havelimitations as additives to therapeutics including xenobioticmetabolism, irritation or toxicity, or high cost. Contemplated for usewith the covalently modified peptides and/or proteins described hereinare excipients that are effective, non-irritating and non-toxic, do notrequire xenobiotic metabolism since they are comprised of the naturalsugars, fatty acids, or long chain alcohols, and which may also be usedto minimize aggregation in aqueous solutions or upon aqueousreconstitution of dried peptide and/or protein formulations in situ byphysiologic aqueous reconstitution by aqueous body fluids such as plasmaor saliva.

Other formulation components could include buffers and physiologicalsalts, non-toxic protease inhibitors such as aprotinin and soybeantrypsin inhibitor, alpha-1-antitrypsin, and protease-inactivatingmonoclonal antibodies, among others. Buffers could include organics suchas acetate, citrate, gluconate, fumarate, malate, polylysine,polyglutamate, chitosan, dextran sulfate, etc, or inorganics such asphosphate, and sulfate. Such formulations may additionally contain smallconcentrations of bacteriostatic agents like benzyl alcohol, and thelike.

Formulations suitable for intranasal administration also comprisesolutions or suspensions of the modified peptides and/or proteinproducts described herein in an acceptable evaporating solvents such ashydrofluoroalkanes. Such formulations are suitable for administrationfrom metered dose inhalers (MDI) and have advantages of lack of movementfrom site of administration, low irritation and absence of need forsterilization. Such formulations may also contain acceptable excipientsor bulking agents such as submicron anhydrous α-lactose.

In yet another aspect, the covalently modified peptides and/or proteinsdescribed herein exhibit increased shelf-life. As used herein, thephrase “shelf life” is broadly described as the length of time a productmay be stored without becoming unsuitable for use or consumption. The“shelf life” of the composition described herein, can also indicate thelength of time that corresponds to a tolerable loss in quality of thecomposition. The compositional shelf life as used herein isdistinguished from an expiration date; “shelf life” relates to thequality of the composition described herein, whereas “expiration date”relates more to manufacturing and testing requirements of thecomposition. For example, a composition that has passed its “expirationdate” may still be safe and effective, but optimal quality is no longerguaranteed by the manufacturer.

Dosing

The covalently modified peptides and/or proteins described herein may beadministered in any amount to impart beneficial therapeutic effect in anumber of disease states. In some embodiments, covalently modifiedpeptides and/or proteins described herein are useful in the treatment ofinflammation. In an embodiment, compounds presented herein impartbeneficial activity in the modulation of post-operative or chronic pain.In an embodiment, the peptides are administered to a patient atconcentrations higher or lower than that of other forms of treatmentwhich modulate pain. In yet another embodiment, the peptides areadministered with other compounds to produce synergistic therapeuticeffects.

Representative delivery regimens include oral, transmucosaladministration, parenteral (including subcutaneous, intraperitoneal,intramuscular and intravenous injection), rectal, buccal (includingsublingual), transdermal, inhalation, ocular and transmucosal (includingintranasal) modes of administration. An attractive and widely usedmethod for delivery of peptides entails subcutaneous injection of acontrolled-release injectable formulation. In some embodiments,covalently modified peptides and/or proteins described herein are usefulfor subcutaneous, intranasal and inhalation administration. Moreover,depending on the condition being treated, these therapeutic compositionsare administered systemically or locally. Techniques for formulation andadministration may be found in the latest edition of “Remington'sPharmaceutical Sciences” (Mack Publishing Co, Easton Pa.).

The selection of the exact dose and composition and the most appropriatedelivery regimen will be influenced by, inter alia, the pharmacologicalproperties of the selected peptide, the nature and severity of thecondition being treated, and the physical condition and mental acuity ofthe recipient. Additionally, the route of administration will result indifferential amounts of absorbed material. Bioavailabilities foradministration of peptides through different routes are particularlyvariable, with amounts from less than 1% to near 100% being seen.Typically, bioavailability from routes other than intravenous,intraperitoneal or subcutaneous injection are 50% or less.

In general, covalently modified peptides and/or proteins describedherein, or salts thereof, are administered in amounts between about 0.1and 1000 μg/kg body weight per day, or between about 0.1 to about 100μg/kg body weight per day, by subcutaneous injection. For a 50 kg humanfemale subject, the daily dose of active ingredient is from about 5 toabout 5000 μg, or from about 5 to about 5000 μg by subcutaneousinjection. Different doses will be needed, depending on the route ofadministration, the compound potency, the pharmacokinetic profile andthe applicable bioavailability observed. By inhalation, the daily doseis from 1000 to about 20,000 μg, twice daily. In other mammals, such ashorses, dogs, and cattle, higher doses may be required. This dosage maybe delivered in a conventional pharmaceutical composition by a singleadministration, by multiple applications, or via controlled release, asneeded to achieve the most effective results.

Pharmaceutically acceptable salts retain the desired biological activityof the parent peptide without toxic side effects. Examples of such saltsare (a) acid addition salts formed with inorganic acids, for examplehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid and the like; and salts formed with organic acids such as,for example, acetic acid, trifluoroacetic acid, tartaric acid, succinicacid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acids, naphthalene disulfonicacids, polygalacturonic acid and the like; (b) base addition salts orcomplexes formed with polyvalent metal cations such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium,and the like; or with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine; or (c) combinations of(a) and (b), e.g., a zinc tannate salt and the like.

Also contemplated, in some embodiments, are pharmaceutical compositionscomprising as an active ingredient covalently modified peptides and/orproteins described herein, or pharmaceutically acceptable salt thereof,in admixture with a pharmaceutically acceptable, non-toxic carrier. Asmentioned above, such compositions may be prepared for parenteral(subcutaneous, intramuscular or intravenous) administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for intranasal administration, particularly in the form of powders,nasal drops, evaporating solutions or aerosols; for inhalation,particularly in the form of liquid solutions or dry powders withexcipients, defined broadly; and for rectal or transdermaladministration.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985), incorporated herein by reference. Formulations for parenteraladministration may contain as excipients sterile water or saline,alkylene glycols such as propylene glycol, polyalkylene glycols such aspolyethylene glycol, saccharides, oils of vegetable origin, hydrogenatednaphthalenes, serum albumin nanoparticles (as used in Abraxane™,American Pharmaceutical Partners, Inc. Schaumburg Ill.), and the like.For oral administration, the formulation can be enhanced by the additionof bile salts or acylcarnitines. Formulations for nasal administrationmay be solid or solutions in evaporating solvents such ashydrofluorocarbons, and may contain excipients for stabilization, forexample, saccharides, surfactants, submicron anhydrous α-lactose ordextran, or may be aqueous or oily solutions for use in the form ofnasal drops or metered spray. For buccal administration typicalexcipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

When formulated for nasal administration, the absorption across thenasal mucous membrane may be further enhanced by surfactants, such asfor example, glycocholic acid, cholic acid, taurocholic acid, ethocholicacid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid,glycodeoxycholic acid, cyclodextrins and the like in an amount in therange between about 0.1 and 15 weight percent, between about 0.5 and 4weight percent, or about 2 weight percent. An additional class ofabsorption enhancers reported to exhibit greater efficacy with decreasedirritation is the class of alkyl maltosides, such as tetradecylmaltoside(Arnold, J. J., et al. (2004) J Pharm Sci 93: 2205-2213, Ahsan, F., etal. (2001) Pharm Res 18: 1742-1746) and references therein, all of whichare hereby incorporated by reference.

When formulated for delivery by inhalation, a number of formulationsoffer advantages. Adsorption of the active peptide to readily dispersedsolids such as diketopiperazines (for example Technosphere particles;(Pfutzner, A. and Forst, T. (2005) Expert Opin Drug Deliv 2: 1097-1106)or similar structures gives a formulation which results in a rapidinitial uptake of the therapeutic agent. Lyophilized powders, especiallyglassy particles, containing the active peptide and an excipient areuseful for delivery to the lung with good bioavailability, for example,sec Exubcra® (inhaled insulin by Pfizer and Aventis PharmaceuticalsInc.). Additional systems for delivery of peptides by inhalation aredescribed (Mandal, T. K., Am. J. Health Syst. Pharm. 62: 1359-64(2005)).

Delivery of covalently modified peptides and/or proteins describedherein to a subject over prolonged periods of time, for example, forperiods of one week to one year, may be accomplished by a singleadministration of a controlled release system containing sufficientactive ingredient for the desired release period. Various controlledrelease systems, such as monolithic or reservoir-type microcapsules,depot implants, polymeric hydrogels, osmotic pumps, vesicles, micelles,liposomes, transdermal patches, iontophoretic devices and alternativeinjectable dosage forms may be utilized for this purpose. Controlledrelease excipients have also been developed for twice weekly or weeklyadministrations, for example, a protected graft copolymer system(Castillo, G. M., et al. (2012) Pharm Res 29: 306-18) can be used forhydrophobic or hydrophobically modified peptides such as those of theinvention. Localization at the site to which delivery of the activeingredient is desired is an additional feature of some controlledrelease devices, which may prove beneficial in the treatment of certaindisorders.

One form of controlled release formulation contains the peptide or itssalt dispersed or encapsulated in a slowly degrading, non-toxic,non-antigenic polymer such as copoly(lactic/glycolic) acid, as describedin the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat. No.4,675,189, incorporated by reference herein. The compounds, or theirsalts, may also be formulated in cholesterol or other lipid matrixpellets, or silastomer matrix implants. Additional slow release, depotimplant or injectable formulations will be apparent to the skilledartisan. See, for example, Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., New York,1978, and R. W. Baker, Controlled Release of Biologically Active Agents,John Wiley & Sons, New York, 1987.

An additional form of controlled-release formulation comprises asolution of a biodegradable polymer, such as copoly(lactic/glycolicacid) or block copolymers of lactic acid and PEG, is bioacceptablesolvent, which is injected subcutaneously or intramuscularly to achievea depot formulation. Mixing of the peptides described herein with such apolymeric formulation is suitable to achieve very long duration ofaction formulations.

As used herein, “therapeutically effective amount” is interchangeablewith “effective amount” for purposes herein, and is determined by suchconsiderations as are known in the art. The amount must be effective toachieve a desired drug-mediated effect in the treated subjects sufferingfrom the disease thereof. A therapeutically effective amount alsoincludes, but is not limited to, appropriate measures selected by thoseskilled in the art, for example, improved survival rate, more rapidrecovery, or amelioration, improvement or elimination of symptoms, orother acceptable biomarkers or surrogate markers.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular subject in need of treatment maybe varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andduration of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and the hostundergoing therapy.

The dosing method(s) includes all aspects of the compositions describedherein including but not limited to compositions which reduce oreliminate immunogenicity of peptide and/or protein drugs, arenon-irritating, have anti-bacterial or anti-fungal activity, haveincreased stability or bioavailability of a drug, decrease thebioavailability variance of that drug, avoid first pass liver clearanceand reduce or eliminate any adverse effects. As used herein, the term“immunogenicity” is the ability of a particular substance or compositionor agent to provoke an immunological response. The immunogenicity of thecovalently modified peptides and/or proteins described herein isconfirmed by methods known in the art.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application is specifically andindividually indicated to be incorporated by reference.

The covalently modified peptides and/or proteins described herein andthe reagents for the synthesis thereof are more particularly describedin the following examples which are intended as illustrative only sincenumerous modifications and variations therein will be apparent to thoseof ordinary skill in the art.

EXAMPLES Example 1: Reagents—N-α-Fmoc, N-ε-(1-octylβ-D-glucuronide-6-yl)-L-lysine

In an oven-dried 250 mL Erlenmeyer flask is placed 1-octylβ-D-glucuronic acid (Carbosynth Ltd., 3.06 g, 10 mmol), 50 mL anhydrousDMF, and anhydrous 1-hydroxybenzotriazole (1.62 g, 12 mmol). A chilled(4° C.) solution of N, N′-dicyclohexylcarbodiimide (2.48 g, 12 mmol) in50 mL of DMF is added, with stirring, and the reaction is allowed toproceed for 5 min. The copious white precipitate of N,N′-dicyclohexylurea is filtered on a fritted glass funnel and thefiltrate is added to a solution of N-α-Fmoc-L-lysine (3.68 g, 10 mmol)in 25 ml anhydrous DMF. The reaction is allowed to proceed for 25 minwith warming to room temp or until the ninhydrin color is very faint.The reaction mixture is filtered, stripped to dryness and crystallizedfrom McOH/Et₂O by dissolution in MeOH and slow dilution to the cloudpoint with Et₂O, followed by refrigeration. Further purification can beachieved by silica gel chromatography using a solvent gradient fromEtOAc to EtOAc/EtOH/AcOH.

In a similar manner, but substituting N-α-Boc-L-lysine is obtainedN-α-Boc,N-ε-(1-octyl β-D-glucuronide-6-yl)-L-lysine, suitable forN-terminal incorporation and cleavage to a free N-Terminus. In a similarmanner, but substituting N-α-Ac-L-lysine is obtained N-α-Ac,N-s-(1-octylβ-D-glucuronide-6-yl)-L-lysine, suitable for incorporation at theN-terminus of a peptide with a blocked N-terminus. In a similar manner,but substituting the appropriate amount of N-α-Fmoc-L-ornithine isobtained N-α-Fmoc,N-δ-(1-octyl β-D-glucuronide-6-yl)-L-ornithine. In asimilar manner but substituting other N-mono-protected diamino acids oneobtains the corresponding reagents. Alternatively, use of a transientMe₃Si ester protecting group during the coupling and withoutpreactivation of the 1-octyl β-D-glucuronic acid provides a facile routeto the formation of the reagents. The transient Me₃Si ester is producedby reaction of the Fmoc-Lys-OH with an equimolar amount ofN,O-bis(trimethylsilyl)acetamide in dichloromethane (CH₂Cl₂). Theorganic layer contains the desired reagent as a solution in CH₂Cl₂ readyfor coupling with the 1-alkyl glucuronide as above. The filteredreaction mixture is washed with aqueous NaHSO₄ to hydrolyze the Me₃Siester, dried over MgSO₄ and solvent is removed.

Similarly, but using peracetyl or perbenzoyl 1-octyl β-D-glucuronic acidone obtains the Ac, or Bz protected form of the reagents (e.g.2,3,4-triacetyl 1-octyl β-D-glucuronic acid, and the like, formed bytreatment with Ac₂O). Such reagents have increased stability during acidcleavage from the resin and are used when instability duringdeprotection is detected, see (Kihlberg, J., et al. (1997) MethodsEnzymol 289: 221-245) and references therein. Final deprotection of suchproducts is carried out by base-catalyzed transesterification aftercleavage, by use of MeOH/NH₃, MeOH/NaOMe, MeOH/NH₂NH₂, as describedabove.

Example 2: Synthetic Peptide Analogs

In general, peptide synthesis methods involve the sequential addition ofprotected amino acids to a growing peptide chain. Normally, either theamino or carboxyl group of the first amino acid and any reactive sidechain group are protected. This protected amino acid is then eitherattached to an inert solid support, or utilized in solution, and thenext amino acid in the sequence, also suitably protected, is added underconditions amenable to formation of the amide linkage. After all thedesired amino acids have been linked in the proper sequence, protectinggroups and any solid support are removed to afford the crude peptide.The peptide is desalted and purified chromatographically.

A preferred method of preparing the analogs of the physiologicallyactive truncated peptides, having fewer than about fifty amino acids,involves solid phase peptide synthesis. In this method the α-amino (Nα)functions and any reactive side chains are protected by acid- orbase-sensitive groups. The protecting group should be stable to theconditions of peptide linkage formation, while being readily removablewithout affecting the extant peptide chain. Suitable α-amino protectinggroups include, but are not limited to t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl,biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc),isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxy-carbonyl,o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl,9-fluorenyl-methoxycarbonyl (Fmoc) and the like, preferably Boc or morepreferably, Fmoc. Suitable side chain protecting groups include, but arenot limited to: acetyl, benzyl (Bzl), benzyloxymethyl (Bom), Boc,t-butyl, o-bromobenzyloxycarbonyl, t-butyl, t-butyldimethylsilyl,2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl,isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl (Tos),2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), trimethylsilyland trityl. A preferred Nα-protecting group for synthesis of thecompounds is the Fmoc group. Preferred side chain protecting groups areO-t-Butyl group for Glu, Tyr, Thr, Asp and Ser; Boc group for Lys andTrp side chains: Pbf group for Arg; Trt group for Asn, Gln, and His. Forselective modification of a Lys residue, orthogonal protection with aprotecting group not removed by reagents that cleave the Fmoc or t-butylbased protecting groups is preferred. Preferred examples formodification of the Lys side chain include, but are not limited to,those removed by hydrazine but not piperidine; for example1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) or1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) andallyloxycarbonyl (Alloc). The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde)protecting group scheme is preferred in cases where side chain lactamformation is desired (Houston, M. E., Jr., et al. (1995) J Pept Sci 1:274-282; Murage, E. N., et al. (2010) J Med Chem), since in this caseFmoc-Glu(O-Allyl) and Fmoc-Lys(Alloc) can be incorporated and used toprovide transient protection, then deprotected for lactam formationwhile the Lys(Dde) protecting group remains for later removal andreaction with the functionalized surfactant.

The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde) protecting group scheme ispreferred in cases where side chain lactam formation is desired(Houston, M. E., Jr., et al. (1995) J Pept Sci 1: 274-282; Murage, E.N., et al. (2010) J Med Chem), since in this case Fmoc-Glu(O-Allyl) andFmoc-Lys(Alloc) can be incorporated and used to provide transientprotection, then deprotected for lactam formation while the Lys(Dde)protecting group remains for later removal and reaction with thefunctionalized surfactant.

In solid phase synthesis, the C-terminal amino acid is first attached toa suitable resin support. Suitable resin supports are those materialswhich are inert to the reagents and reaction conditions of the stepwisecondensation and deprotection reactions, as well as being insoluble inthe media used. Examples of commercially available resins includestyrene/divinylbenzene resins modified with a reactive group, e.g.,chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylatedco-poly-(styrene-divinylbenzene), and the like. Benzylated,hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred for thepreparation of peptide acids. When the C-terminus of the compound is anamide, a preferred resin isp-methylbenzhydrylamino-co-poly(styrene-divinylbenzene) resin, a 2,4dimethoxybenzhydrylamino-based resin (“Rink amide”), and the like. Anespecially preferred support for the synthesis of larger peptides arecommercially available resins containing PEG sequences grafted ontoother polymeric matricies, such as the Rink Amide-PEG and PAL-PEG-PSresins (Applied Biosystems) or similar resins designed for peptide amidesynthesis using the Fmoc protocol. Thus in certain cases it is desirableto have an amide linkage to a PEG chain. It those cases it is convenientto link an N-Fmoc-amino-PEG-carboxylic acid to the amide forming resinabove (e.g. Rink amide resin and the like). The first amino acid of thechain can be coupled as an N-Fmoc-amino acid to the amino function ofthe PEG chain. Final deprotection will yield the desiredPeptide-NH-PEG-CO—NH₂ product.

Attachment to the PAM resin may be accomplished by reaction of the Nαprotected amino acid, for example the Boc-amino acid, as its ammonium,cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene,tetramethylammonium, or similar salt in ethanol, acetonitrile,N,N-dimethylformamide (DMF), and the like, preferably the cesium salt inDMF, with the resin at an elevated temperature, for example betweenabout 40° and 60° C., preferably about 50° C., for from about 12 to 72hours, preferably about 48 hours. This will eventually yield the peptideacid product following acid cleavage or an amide following aminolysis.

The Nα-Boc-amino acid may be attached to the benzhydrylamine resin bymeans of, for example, an N,N′-diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HOBt) mediated coupling for from about 2to about 24 hours, preferably about 2 hours at a temperature of betweenabout 10° and 50° C., preferably 25° C. in a solvent such as CH₂Cl₂ orDMF, preferably CH₂Cl₂.

For Boc-based protocols, the successive coupling of protected aminoacids may be carried out by methods well known in the art, typically inan automated peptide synthesizer. Following neutralization withtriethylamine, N,N-di-isopropylethylamine (DIEA), N-methylmorpholine(NMM), collidine, or similar base, each protected amino acid isintroduced in approximately about 1.5 to 2.5 fold molar excess and thecoupling carried out in an inert, nonaqueous, polar solvent such asCH₂Cl₂, DMF, N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), ormixtures thereof, preferably in dichloromethane at ambient temperature.For Fmoc-based protocols no acid is used for deprotection but a base,preferably DIEA or NMM, is usually incorporated into the couplingmixture. Couplings are typically done in DMF, NMP, DMA or mixedsolvents, preferably DMF. Representative coupling agents areN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropyl-carbodiimide (DIC)or other carbodiimide, either alone or in the presence of HOBt, O-acylureas, benzotriazol-1-yl-oxytris(pyrrolidino)phosphoniumhexafluorophosphate (PyBop), N-hydroxysuccinimide, otherN-hydroxyimides, or oximes. Alternatively, protected amino acid activeesters (e.g. p-nitrophenyl, pentafluorophenyl and the like) orsymmetrical anhydrides may be used. Preferred coupling agents are of theaminium/uronium (alternative nomenclatures used by suppliers) class suchas 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HBTU),O-(7-azabenzotraiazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),2-(6-Chloro-1H-benzotraiazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU), and the like.

A preferred method of attachment to the Fmoc-PAL-PEG-PS resin may beaccomplished by deprotection of the resin linker with 20% piperidine inDMF, followed by reaction of the N-α-Fmoc protected amino acid, about a5 fold molar excess of the N-α-Fmoc-amino acid, using HBTU:di-isopropylethylamine (DIEA) (1:2) in DMF in a microwave-assistedpeptide synthesizer with a 5 min, 750 max coupling cycle.

For this Fmoc-based protocol in the microwave-assisted peptidesynthesizer, the N-α-Fmoc amino acid protecting groups are removed with20% piperidine in DMF containing 0.1M 1-hydroxybenzotriazole (HOBt), ina double deprotection protocol for 30 sec and then for 3 min with atemperature maximum set at 75° C. HOBt is added to the deprotectionsolution to reduce aspartamide formation. Coupling of the next aminoacid then employs a five-fold molar excess using HBTU:DIEA (1:2) with a5 min, 750 max. double-coupling cycle.

At the end of the solid phase synthesis the fully protected peptide isremoved from the resin. When the linkage to the resin support is of thebenzyl ester type, cleavage may be effected by means of aminolysis withan alkylamine or fluoroalkylamine for peptides with an alkylamideC-terminus, or by ammonolysis with, for example, ammonia/methanol orammonia/ethanol for peptides with an unsubstituted amide C-terminus, ata temperature between about −10° and 500° C., preferably about 25° C.,for between about 12 and 24 hours, preferably about 18 hours. Peptideswith a hydroxy C-terminus may be cleaved by HF or other strongly acidicdeprotection regimen or by saponification. Alternatively, the peptidemay be removed from the resin by transesterification, e.g., withmethanol, followed by aminolysis or saponification. The protectedpeptide may be purified by silica gel or reverse-phase HPLC.

The side chain protecting groups may be removed from the peptide bytreating the aminolysis product with, for example, anhydrous liquidhydrogen fluoride in the presence of anisole or other carbonium ionscavenger, treatment with hydrogen fluoride/pyridine complex, treatmentwith tris(trifluoroacetyl)boron and trifluoroacetic acid, by reductionwith hydrogen and palladium on carbon or polyvinylpyrrolidone, or byreduction with sodium in liquid ammonia, preferably with liquid hydrogenfluoride and anisole at a temperature between about −10° and +10° C.,preferably at about 0° C., for between about 15 minutes and 2 hours,preferably about 1.5 hours.

For peptides on the benzhydrylamine type resins, the resin cleavage anddeprotection steps may be combined in a single step utilizing liquidhydrogen fluoride and anisole as described above or preferably throughthe use of milder cleavage cocktails. For example, for the PAL-PEG-PSresin, a preferred method is through the use of a double deprotectionprotocol in the microwave-assisted peptide synthesizer using one of themild cleavage cocktails known in the art, such asTFA/water/tri-iso-propylsilane/3,6-dioxa-1,8-octanedithiol (DODT)(92.5/2.5/2.5/2.5) for 18 min at 38° C. each time. Cleavage of alkylglycoside containing materials have shown survival of the alkylglycoside linkage using protocols with TFA/water ratios in the 9/1 to19/1 range. A typical cocktail is 94% TFA: 2% EDT; 2% H₂O; 2% TIS.Typically the fully deprotected product is precipitated and washed withcold (−70° to 4° C.) Et₂O, dissolved in deionized water and lyophilized.

The peptide solution may be desalted (e.g. with BioRad AG-3® anionexchange resin) and the peptide purified by a sequence ofchromatographic steps employing any or all of the following types: ionexchange on a weakly basic resin in the acetate form; hydrophobicadsorption chromatography on underivatizedco-poly(styrene-divinylbenzene), e.g. Amberlite® XAD; silica geladsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g. on Sephadex®G-25; counter-current distribution; supercritical fluid chromatography;or HPLC, especially reversed-phase HPLC on octyl- oroctadecylsilylsilica (ODS) bonded phase column packing.

Also provided herein are processes for preparing covalently modifiedpeptides and/or proteins described herein and pharmaceuticallyacceptable salts thereof, which processes comprise sequentiallycondensing protected amino acids on a suitable resin support, removingthe protecting groups and resin support, and purifying the product, toafford analogs of the physiologically active truncated homologs andanalogs of the covalently modified peptides and/or proteins describedherein. In some embodiments, covalently modified peptides and/orproteins described herein incorporate alkyl glycoside modifications asdefined above. Another aspect relates to processes for preparingcovalently modified peptides and/or proteins described herein andpharmaceutically acceptable salts thereof, which processes comprise theuse of microwave-assisted solid phase synthesis-based processes orstandard peptide synthesis protocols to sequentially condense protectedamino acids on a suitable resin support, removing the protecting groupsand resin support, and purifying the product, to afford analogs of thephysiologically active peptides, as defined above.

Example 3. General Oxidation Method for Uronic Acids

To a solution of 1-dodecyl β-D-glucopyranoside (Carbosynth) [2.0 g, 5.74mmol] in 20 mL of acetonitrile and 20 mL of DI water was added(diacetoxyiodo)benzene (Fluka) [4.4 g, 13.7 mmol] and TEMPO(SigmaAldrich) [0.180 g, 1.15 mmol]. The resulting mixture was stirredat room temperature for 20 h. The reaction mixture was diluted withwater and lyophilized to dryness to give 1.52 g (crude yield 73.1%) ofthe crude product, 1-dodecyl β-D-glucuronic acid, as a white powder,which was used directly for the solid phase synthesis without furtherpurification. This product was previously prepared by an alternativeprocess using NaOCl as oxidant, as described in the specification, andalso has been used for longer alkyl groups. In a similar manner areprepared the desired alkyl saccharide uronic acids used to make theproducts and reagents described herein.

In a like manner, but using the corresponding 1-tetradecyl, 1-hexadecyl,and 1-octadecyl β-D-glucopyranosides (purchased from Anatrace, Maumee,Ohio) were prepared the desired 1-alkyl saccharide uronic acids whichwere used to make the products and reagents described herein.

Example 4: Preparation of Analog EU-A387

A sample ofFmoc-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Bip-Ser-Lys-Tyr-Leu-Glu-Ser-Lys(Alloc)-Rinkamide resin was prepared by sequential addition of N-alpha-Fmocprotected amino acids as described in Example 1 and deprotected on theLys-N-epsilon position by incubation with Pd(PPh₃)₄ (0.5 eq) and DMBA(20 eq) in DMF/CH₂Cl₂ (1:1) overnight in the dark at room temperature.Following washing by DMF/CH₂Cl₂, the Lys side chain was acylated with1′-dodecyl β-D-glucuronic acid in DMF/CH₂Cl₂ through the use ofDIC/HOBt. Completion of the coupling was checked by ninhydrin and theproduct was washed extensively with CH₂Cl₂.

The product resin is submitted to final deprotection and cleavage fromthe resin by treatment with the cleavage cocktail (94% TFA: 2% EDT; 2%H₂O; 2% TIS) for a period of 240 min at room temperature. The mixturewas treated with Et₂O, to precipitate the product and washed extensivelywith Et₂O to yield the crude title peptide product after drying invacuo.

Purification is carried out in two batches by reversed phase (C18) hplc.The crude peptide was loaded on a 4.1×25 cm hplc column at a flow rateof 15 mL/min (15% organic modifier; acetic acid buffer) and eluted witha gradient from 15-45% buffer B in 60 min at 50° C. The product fractionis lyophilized to yield the title product peptide with a purity 98.03%by analytical hplc (18.6 min; 30-60% CH₃CN in 0.1% TFA)/massspectrometry (M+1 peak=2382.14).

The corresponding 1-methyl and 1-octyl analogs of the title compound areprepared in a similar manner, but using the reagents 1′-methylβ-D-glucuronic acid and 1′-octyl β-D-glucuronic acid (Carbosynth). Thecorresponding 1-decyl, 1-dodecyl, 1-tetradecyl, 1-hexadecyl, -octadecyland 1-eicosyl analogs are prepared using the corresponding glucoronicacids, prepared as described above. Alternatively, the 1-alkylglucuronyl, or other uronic acylated analogs, may be prepared by initialpurification of the deprotected or partially deprotected peptidefollowed by acylation by the desired uronic acid reagent.

Analysis was done by HPLC/mass spectrometry in positive ion mode usingthe eluent gradients given in the table below.

Compound Molecular Wt Molecular Wt HPLC Name Expected found (min;elution) EU-A387 2379.66 2380.14 18.6 [b] EU-A388 2393.69 2393.74 16.0[a] EU-A391 2317.62 2318.26 11.2 |b] EU-A455 2988.36 2988.00 11.5 [b]EU-A474 2570.86 2570.54 11.3 [b] EU-A478 2459.75 2459.74 11.1 [b]EU-A484 2544.86 2545.06 9.6 [b] EU-A501 2904.2 2903.34 7.9 [b] EU-A5022776.07 2776.14 8.0 [b] EU-A503 2704.98 2704.40 8.0 [b] EU-A504 2548.802548.00 9.1 [b] EU-A505 2392.61 2392.40 10.5 [b] EU-A506 2305.53 2305.0610.7 [b] EU-A507 3763.23 3762.66 9.0 [b] EU-A521 2303.56 2303.60 8.2 [c]EU-A522 2315.60 2315.60 14.2 [d] EU-A523 2615.94 2616.00 8.1 [b] EU-A5242459.75 2459.74 12.7 [d] EU-A525 2459.75 2459.06 6.0 [c] EU-A526 2473.752473.60 12.7 [d] EU-A527 2390.64 2390.40 14.6 [d] EU-A529 2546.832546.80 9.5 [b] EU-A531 2546.83 2546.80 9.5 [b] EU-A532 2559.00 2558.669.6 [b] EU-A533 2560.96 2560.66 9.5 [b] EU-A534 2544.99 2544.94 9.7 [b]EU-A535 2573.05 2574.00 12.0 [b] EU-A536 2602.96 2603.46 14.3 [b]EU-A538 2516.99 2516.40 10.3 [b] EU-A539 2657.20 2656.80 10.8 [b]EU-A540 2685.20 2684.94 9.8 [c] EU-A541 2713.20 2712.80 13.0 [c] EU-A5442631.94 2632.26 10.8 [b] EU-A546 EU-A549 2388.67 2388.66 6.3 [e] EU-A5512444.67 2445.20 11.4 [e] EU-A552 EU-A554 2560.86 2560.40 10.3 [c]EU-A556 2616.86 2616.40 11.7 [e] EU-A560 2570.86 2571.06 8.3 [c] EU-A5622626.86 2626.66 9.9 [e] EU-A563 EU-A565 2542.80 2542.54 9.5 [c] EU-A5672598.80 2599.06 12.0 [c] EU-A568 HPLC gradients in 0.1% TFA [a] 35 to65% CH₃CN over 30 min. [b] 30 to 60% CH₃CN over 20 min. [c] 35 to 65%CH₃CN over 20 min. [d] 25 to 55% CH₃CN over 20 min. [e] 40 to 70% CH₃CNover 20 min. HPLC on Phenomenex Luna C18 5 micron 250 × 4.6 mm.

Example 5: Cellular Assay of the Compounds

Compounds were weighed precisely in an amount of approximately 1 mg andassayed in standard cellular assays (Cerep SA). The readout is theamount of cAMP generated in the cells treated with the test compounds,in either agonist or antagonist mode. The assay used was the stimulationof cAMP levels in the glucagon and GLP-1 cellular assays. The assays aredescribed in Chicchi, G. G., et al. (1997) J Biol Chem 272: 7765-7769and Runge, S., et al. (2003) Br J Pharmacol 138: 787-794.

For compound EU-A391 the GLCR cellular response does not change and theGLP1R cellular response rises steeply with and EC50 of 420 nM

EC₅₀ EC₅₀ GLP-1 R glucagon R Compound Structure (nM) (nM) EU-A3911-dodecyl 420 n.c. EU-A455 1-dodecyl 59  770 EU-A474 1-dodecyl 3000 n.c.EU-A478 1-dodecyl n.c. n.c. EU-A484 1-dodecyl n.c. n.c. EU-A5011-dodecyl 20000 12000  EU-A502 1-dodecyl 9400 n.c. EU-A503 1-dodecyln.c. n.c. EU-A504 1-dodecyl 3100 1100 EU-A505 1-dodecyl 8500 6100EU-A506 1-dodecyl 4600 1300 EU-A507 1-dodecyl 18   1 EU-A521 1-dodecyln.c. n.c. EU-A522 1-dodecyl n.c. 9000 EU-A523 1-dodecyl n.c. n.c.EU-A524 1-dodecyl n.c. n.c. EU-A525 1-dodecyl n.c. n.c. EU-A5261-dodecyl n.c. n.c. EU-A527 1-dodecyl n.c. 5000 EU-A529 1-dodecyl n.c.7000 EU-A531 1-dodecyl 2100 1100 EU-A532 1-dodecyl 5000 2600 EU-A5331-dodecyl 770  780 EU-A534 1-dodecyl 290 1900 EU-A535 1-tetradecyl §48002100 EU-A536 1-hexadecyl >10000 4400 EU-A538 1-dodecyl 270 n.c. EU-A5391-dodecyl 860 2300 EU-A540 1-tetradecyl n.c. 8800 EU-A541 1-hexadecyl800 5000 n.c. means EC₅₀ not calculable §means superagonist

Example 6: In Vivo Assay of Compounds

Sixty (60) diet induced obese C57BL/6J male mice are received from JAXlabs at 14 wks of age. The mice are ear notched for identification andhoused in individually and positively ventilated polycarbonate cageswith HEPA filtered air at density of one mouse per cage. The animal roomis lighted entirely with artificial fluorescent lighting, with acontrolled 12 h light/dark cycle. The normal temperature and relativehumidity ranges in the animal rooms are 22±4° C. and 50±15%,respectively. Filtered tap water, acidified to a pH of 2.8 to 3.1, andhigh fat diet (60 kcal %) are provided ad libitum.

Following a 2 week acclimation, 40 mice are chosen based on desired bodyweight range and mice are randomized into groups (n=10) as below.Group 1. Vehicle treated; Group 2. Low dose test cmpd; Group 3. Mid dosetest cmpd; Group 4. High dose test cmpd. Mice are dosed via SC daily for28 days. Body weights and cage side observations are recorded daily.Food and water intake will be recorded weekly. Mice undergo NMRmeasurements for determining whole body fat and lean composition on days1 (pre dose) and 26. On days 0, 14 and 27, mice are fasted overnight foran oral glucose tolerance test. Next day, the first blood sample iscollected via tail nick (t=0). Mice are then administered a bolus of 1.0g/kg glucose. Blood samples are obtained via tail nick at 5, 30, 60 and120 min after glucose and plasma glucose will be immediately determinedusing a glucometer.

Sacrifice and tissue collection: Mice are sacrificed on day 29. Terminalblood is processed to serum/plasma and aliquots are sent for analysis ofglucose, insulin and lipid profile. Fat tissues are collected, weighedand frozen for analysis. The optimal compound profile shows decreasedglucose excursion in the OGTT, decreased basal insulin secretion, withpotentiated glucose-dependent insulin secretion, decreased weight gain,decreased fat mass but minimal effects on lean mass.

Example 7: Uses of the Compounds

The covalently modified peptides and/or proteins described herein areuseful for the prevention and treatment of a variety of diseases relatedto obesity, the metabolic syndrome, cardiovascular disease and diabetes.Suitably labeled surfactant modified peptides can be used as diagnosticprobes.

Representative delivery regimens include oral, parenteral (includingsubcutaneous, intramuscular and intravenous injection), rectal, buccal(including sublingual), transdermal, inhalation ocular and intranasal.An attractive and widely used method for delivery of peptides entailssubcutaneous injection of a controlled release injectable formulation.Other administration routes for the application of the covalentlymodified peptides and/or proteins described herein are subcutaneous,intranasal and inhalation administration.

Example 8. Pharmaceutical Usage for Treatment of Insulin Resistance

A human patient, with evidence of insulin or metabolic syndrome istreated with EU-A596 by intranasal administration (200 μL) from astandard atomizer used in the art of a solution of the pharmaceuticalagent in physiological saline containing from 0.5 to 10 mg/mL of thepharmaceutical agent and containing standard excipients such as benzylalcohol. The treatment is repeated as necessary for the alleviation ofsymptoms such as obesity, elevated blood glucose and the like. In asimilar manner, a solution of EU-A596, and selected excipients, in anevaporating solvent containing such as a hydrofluoroalkane isadministered intranasally by metered dose inhaler (MDI) as needed toreduce insulin resistance. The effect of treatment is determined usingstandard tests including measurement of blood glucose levels, Body MassIndex, and/or body weight and/or measurement of waist to hip ratios.

In a similar manner, administration of an adjusted amount bytransbuccal, intravaginal, inhalation, subcutaneous, intravenous,intraocular, or oral routes is tested to determine level of stimulationof GLP1R and/or GLCR on cells in the body and to determine therapeuticeffects.

SEQUENCES

The specification provides sequences for SEQ. ID. Nos. 1-3 and SEQ. ID.Nos. 318-343. Additionally, Table 1 of FIG. 1 provides SEQ. ID Numbersfor compounds EU-A300 to EU-A425 having SEQ. ID. NOs. 4-129respectively, as shown in Table 1 of FIG. 1. Compounds in Table 1 ofFIG. 1, and their respective SEQ. ID. NOs. shown in Table 1 of FIG. 1are hereby incorporated into the specification as filed. Additionally,Table 2 of FIG. 2 provides SEQ. ID Numbers for compounds EU-A426 toEU-599 having SEQ. ID. NOs. 130-317 respectively, as shown in Table 2 ofFIG. 2. Compounds in Table 2 of FIG. 2, and their respective SEQ. ID.NOs. shown in Table 2 of FIG. 2 are hereby incorporated into thespecification as filed.

1. A peptide product comprising a surfactant X, covalently attached to apeptide, the peptide comprising a linker amino acid U and at least aminoacid residues aa₁-aa₂₀ of SEQ. ID. NO. 1:

wherein the surfactant X is a group of Formula I:

wherein: R^(1a) is independently, at each occurrence, a bond, H, asubstituted or unsubstituted C₁-C₃₀ alkyl group, a substituted orunsubstituted alkoxyaryl group, a substituted or unsubstituted aralkylgroup, or a steroid nucleus containing moiety; R^(1b), R^(1c), andR^(1d) are each, independently at each occurrence, a bond, H, asubstituted or unsubstituted C₁-C₃₀ alkyl group, a substituted orunsubstituted alkoxyaryl group, or a substituted or unsubstitutedaralkyl group; W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,—(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C—S)—NH—, or —CH₂—S—; W² is—O—, —CH₂— or —S—; R² is a bond to U, n is 1, 2 or 3; and Formula II(SEQ. ID. NO. 1) aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-aa₃₀-aa₃₁-aa₃₂-aa₃₃-aa₃₄- aa₃₅-aa₃₆-aa₃₇-Z

wherein: Z is OH, or —NH—R³, wherein R³ is H, or C1-C₁₂ substituted orunsubstituted alkyl, or a PEG chain of less than 10 Da; aa₁ is His,N—Ac-His, pGlu-His, or N—R³-His; aa₂ is Ser, Ala, Gly, Aib, Ac4c, orAc5c; aa₃ is Gln, or Cit; aa₄ is Gly, or D-Ala; aa₅ is Thr, or Ser; aa₆is Phe, Trp, F2Phe, Me2Phe, or Nal2; aa₇ is Thr, or Ser; aa₈ is Ser, orAsp; aa₉ is Asp, or Glu; aa₁₀ is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO;aa₁₁ is Ser, Asn, or U; aa₁₂ is Lys, Glu, Ser, Arg, or U; aa₁₃ is Tyr,Gln, Cit, or U; aa₁₄ is Leu, Met, Nle, or U; aa₁₅ is Asp, Glu, or U;aa₁₆ is Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or U; aa₁₇ is Arg, hArg,Gln, Glu, Cit, Aib, Ac4c, Ac5c, or U; aa₁₈ is Arg, hArg, Ala, Aib, Ac4c,Ac5c, or U; aa₁₉ is Ala, Val, Aib, Ac4c, Ac5c, or U; aa₂₀ is Gln, Lys,Arg, Cit, Glu, Aib, Ac4c, Ac5c, or U; aa₂₁ is absent or Asp, Glu, Leu,Aib, Ac4c, Ac5c, or U; aa₂₂ is absent or Phe, Trp, Nal2, Aib, Ac4c,Ac5c, or U aa₂₃ is absent or Val, Ile, Aib, Ac4c, Ac5c, or U; aa₂₄ isabsent or Gln, Ala, Glu, Cit, or U; aa₂₅ is absent or Trp, Nal2, or U;aa₂₆ is absent or Leu, or U; aa₂₇ is absent or Met, Val, Nle, Lys, or U;aa₂₈ is absent or Asn, Lys, or U; aa₂₉ is absent or Thr, Gly, Aib, Ac4c,Ac5c, or U; aa₃₀ is absent or Lys, Aib, Ac4c, Ac5c, or U; aa₃₁ is absentor Arg, Aib, Ac4c, Ac5c, or U; aa₃₂ is absent or Asn, Aib, Ac4c, Ac5c,or U; aa₃₃ is absent or Arg, Aib, Ac4c, Ac5c, or U; aa₃₄ is absent orAsn, Aib, Ac4c, Ac5c, or U; aa₃₅ is absent or Asn, Aib, Ac4c, Ac5c, orU; aa₃₆ is absent or Ile, Aib, Ac4c, Ac5C, or U; aa₃₆ is absent or Ala,Aib, Ac4c, Ac5C, or U; aa₃₇ is absent or U; U is a natural or unnaturalamino acid comprising a functional group used for covalent attachment tothe surfactant X; wherein any two of aa₁₁-aa₃₇ are optionally cyclizedthrough their side chains to form a lactam linkage; and provided thatone, or at least one of aa₁₁-aa₃₇ is the linker amino acid U covalentlyattached to X.
 2. The peptide product of claim 1, wherein n is
 1. 3. Apeptide product of claim 1, wherein X has the structure:

wherein: R^(1a) is H, a protecting group, a substituted or unsubstitutedC₁-C₃₀ alkyl group, or a steroid nucleus containing moiety; R^(1b),R^(1c), and R^(1d) are each, independently at each occurrence, H, aprotecting group, or a substituted or unsubstituted C₁-C₃₀ alkyl group;W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—, —(C═O),—(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C—S)—NH—, or —CH₂—S—; W² is —O—, —S—;and, R² is a bond to U.
 4. The peptide product of claim 3, wherein X hasthe structure:


5. (canceled)
 6. The peptide product of claim 3, wherein X has thestructure:

wherein: R^(1a) is H, a protecting group, a substituted or unsubstitutedC₁-C₃₀ alkyl group, or a steroid nucleus containing moiety; R^(1b),R^(1c), and R^(1d) are each, independently at each occurrence, H, aprotecting group, or a substituted or unsubstituted C₁-C₃₀ alkyl group;W¹ is —(C═O); W² is —O—; R² is a bond to U.
 7. The peptide product ofclaim 3, wherein X has the structure:

wherein: R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;R^(1b), R^(1c), and R^(1d) are H; W¹ is —(C═O); W² is —O—; and R² is abond to U.
 8. The peptide product of claim 1, wherein R^(1a) is asubstituted or unsubstituted C₁-C₃₀ alkyl group, a substituted orunsubstituted C₆-C₂₀ alkyl group, or a substituted or unsubstitutedC₁₂-C₂ alkyl group.
 9. (canceled)
 10. (canceled)
 11. The peptide productof claim 1, wherein the surfactant X is a 1-alkyl glycoside classsurfactant.
 12. The peptide product of claim 1, wherein X is comprisedof 1-eicosyl beta-D-glucuronic acid, 1-octadecyl beta-D-glucuronic acid,1-hexadecyl beta-D-glucuronic acid, 1-tetradecylbeta D-glucuronic acid,1-dodecyl beta D-glucuronic acid, 1-decyl beta-D-glucuronic acid,1-octyl beta-D-glucuronic acid, 1-eicosyl beta-D-diglucuronic acid,1-octadecyl beta-D-diglucuronic acid, 1-hexadecyl beta-D-diglucuronicacid, 1-tetradecyl beta-D-diglucuronic acid, 1-dodecylbeta-D-diglucuronic acid, 1-decyl beta-D-diglucuronic acid, 1-octylbeta-D-diglucuronic acid, or functionalized 1-ecosyl beta-D-glucose,1-octadecyl beta-D-glucose, 1-hexadecyl beta-D-glucose, 1-tetradecylbeta-D-glucose, 1-dodecyl beta-D-glucose, 1-decyl beta-D-glucose,1-octyl beta-D-glucose, 1-eicosyl beta-D-maltoside, 1-octadecylbeta-D-maltoside, 1-hexadecyl beta-D-maltoside, 1-dodecylbeta-D-maltoside, 1-decyl beta-D-maltoside, or 1-octyl beta-D-maltoside.13. The peptide product of claim 1, wherein U is selected from Lys, Cys,Orn, or an unnatural amino acid comprising a functional group used forcovalent attachment to the surfactant X. 14-18. (canceled)
 19. Thepeptide product of claim 1, wherein aa₂ is an Aib or Ac4c residue. 20.The peptide product of claim 1, wherein the peptide comprises one ormore Aib residues. 21-24. (canceled)
 25. The peptide product of claim 1,wherein aa₁₆ and aa₂₀ are cyclized to form a lactam linkage. 26-31.(canceled)
 32. A pharmaceutical composition comprising a therapeuticallyeffective amount of a peptide product of claim 1, or acceptable saltthereof, and at least one pharmaceutically acceptable carrier orexcipient.
 33. A method of treating a condition associated with insulinresistance comprising administration of a compound of claim 1 to anindividual in need thereof.
 34. The method of claim 33, wherein theinsulin resistance is diabetes or cardiovascular disease. 35-38.(canceled)
 39. The method of claim 33, wherein the administration ofsaid peptide product causes weight loss. 40-57. (canceled)
 58. Thepeptide product of claim 1, wherein the peptide comprises at leastaa₁-aa₂₆ of SEQ ID NO.
 1. 59. The peptide product of claim 58, whereinaa₁₇ is U(X) and aa₁₆ and aa₂₀ are cyclized to form a lactam linkage.60. The peptide product of claim 59, wherein X has the structure:

wherein: R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;R^(1b), R^(1c), and R^(1d) are H; W¹ is —(C═O); W² is —O—; and R² is abond to U.